Fluorescent compounds

ABSTRACT

The present invention relates to fluorescent dyes. The present invention provides a wide range of fluorescent dyes and kits containing the same, which are applicable for labeling a variety of biomolecules, cells and microorganisms. In one aspect, the invention provides a compound having a maximal fluorescence excitation wavelength, wherein the compound has a structure of Formula II:
 
F—Y=Ψ  Formula II
 
and wherein Z— is a counterion, Y is a bridge unit permitting electron delocalization between F and Ψ, and F is a moiety having the structure:
 
     
       
         
         
             
             
         
       
     
     The present invention also provides various methods of using the fluorescent dyes for research and development, forensic identification, environmental studies, diagnosis, prognosis, and/or treatment of disease conditions.

This application claims the benefit of U.S. Provisional Application Ser.No. 61/013,956, filed on Dec. 14, 2007, which is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Fluorescent dyes are widely used in biological research and medicaldiagnostics. Fluorescent dyes are superior to conventional radioactivematerials because fluorescent dyes are typically sufficiently sensitiveto be detected, less expensive and less toxic. In particular, adiversity of fluorophores with a distinguishable color range has made itmore practical to perform multiplexed assays capable of detectingmultiple biological targets in parallel. The ability to visualizemultiple targets in parallel is often required for delineating thespatial and temporal relationships amongst different biological targetsin vitro and in vivo. In addition, the generation of a wide range offluorescent dyes has opened a new avenue for conducting high-throughputand automated assays, thus dramatically reducing the unit cost perassay. Moreover, the low toxicity of fluorescent dyes provides ease ofhandling in vitro, and also renders it safer for imaging biologicalactivities in vivo.

Despite the various advantages of fluorescent dyes, conventional dyeshave a number of profound limitations. For example, conventionalfluorescent dyes are typically prone to inter-dye quenching, aphenomenon known to diminish the effective brightness of the dyes. It isa common practice to conjugate a given target with multiple dyemolecules in order to maximize the brightness of the labeled target,e.g., a biomolecule such as protein or DNA. For many conventionalfluorescent dyes, the fluorescence intensity of the labeled target isoften not directly proportional to the number of attached dye molecules,but rather less than the predicted intensity due to, e.g., quenchingamongst the multiple dyes attached to the target. Such quenching effectcan be attributed to, in part, the physical interaction amongst theattached dye molecules, which may lead to formation of nonfluorescentdye dimers. Dimer formation may be driven by hydrophobic interaction.Because many traditional fluorescent dyes, such as various rhodaminedyes and cyanine dyes, are highly hydrophobic aromatic compounds, thesecommonly used dyes are particularly prone to forming dimers on labeledbiomolecules. Adding sulfonate groups to a dye has been shown to reducedimer formation. See, e.g., U.S. Pat. Nos. 5,268,486 and 6,977,305,6,130,101 and Panchuk-Voloshina, et al. J. Histochem. Cytochem. 47(9),1179 (1999). However, while sulfonation may reduce dimer formation, italso introduces negative charges into a biomolecule, and thus mayincrease the risk of disrupting the biological activity of the labeledbiomolecule. Furthermore, dyes substituted with sulfonates alone mayexhibit a shorter serum half-life when used in vivo in a subject.

Another limiting factor for conventional fluorescent dyes is the lowfluorescence brightness intrinsic to individual fluorescent groups. Suchproperty is generally determined by the fluorescence quantum yield ofthe fluorescent group. A low fluorescence quantum yield is usually dueto energy transfer from the excited electronic state to the vibrationaland rotational states of the molecule, a process in which the electronicenergy is converted to heat, instead of light. One approach to improvethe fluorescence quantum yield of a fluorescent group is to rigidify thedye structure so that the dye has limited vibrational and rotationalmodes. See, e.g., U.S. Pat. Nos. 5,981,747 and 5,986,093, which describemonomethine cyanine dyes that are rigidified by a two-carbon chain thatlinks the two benzazolium nitrogen atoms. Similarly, in U.S. Pat. No.6,133,445, trimethine cyanine dyes are rigidified by incorporating thebridge moiety into a three fused ring system. The rigidified cyaninedyes all have significantly improved quantum yields compared to thenonrigidified counterpart dyes. However, the improvements in quantumyield are obtained at the expense of other desirable properties. Forexample, because of their relatively complex structures, theserigidified dyes typically take several more steps to synthesize thanregular cyanine dyes, often with low yields. Highly rigidified dyes mayalso show a higher tendency to aggregate on proteins. For example, arigidified cyanine dye has been shown to form dimers even when used at amuch lower degree of labeling on proteins than a nonrigidified cyanine(Cooper, et al. Journal of Fluorescence 14, 145 (2004)). Furthermore,rigidified trimethine cyanine dyes have shown significantly reducedphotostability, compared to regular non-rigidified trimethine cyaninedyes (see, e.g., U.S. Pat. No. 6,133,445).

SUMMARY OF THE INVENTION

Thus there remains a considerable need for improved compositions andmethods that would allow convenient and effective labeling of a widerange of molecules in various applications. The present inventionaddresses this need and provides additional advantages.

Accordingly, the present invention provides fluorescent compounds whichmay have any or all of the following characteristics. In one aspect,labeled biomolecules prepared using fluorescent compounds of theinvention show significantly reduced dimer formation. In other aspects,compounds and labeled biomolecules of the invention show other desirableproperties such as higher water solubility, improved fluorescencequantum yield, improved photostability, relatively simple synthesis,improved specificity of the labeled conjugates, and/or improved in vivohalf-life.

The invention provides a compound of formula I:

wherein

F is a fluorophore;

T is a joining moiety formed of one or more chemical bonds andconnecting three or more distinct moieties, and wherein said joiningmoiety contains about 1-100 atoms;

m and n are independently integers ranging from 0 to 20;

R₁, R₂, and R₃ are each independently (R)_(p)-(L)_(q)-;

each L of R₁, R₂, and R₃ is independently a linking moiety formed of oneor more chemical bond and containing about 1-100 atoms selected suchthat the group is a stable moiety;

each p of R₁, R₂, and R₃ is an integer ranging from 1 to 20;

each q of R₁, R₂, and R₃ is an integer ranging from 0 to 20;

each R of R₁, R₂, and R₃ is independently: i) a reactive group capableof forming a covalent bond upon reacting with a reaction partner; ii) aradical of a water-soluble polymer; iii) an alkyl group, atrifluoroalkyl group, a halogen group, a sulfonyl group, a sulfonategroup, a phosphonate group or a sulfonamido group; or iv) —H; andwherein at least one R of R₁, R₂, and R₃ is a reactive group and atleast one other R of R₁, R₂, and R₃ is a radical of a water-solublepolymer.

In some embodiments, the fluorophore is a xanthene dye, a coumarin dye,a pyrene dye or a cyanine dye. The water soluble group may, for example,be a polyalkylene oxide such as a polyethylene oxide. Alternatively, thewater soluble polymer group may be a carbohydrate or a polypeptide. Thewater soluble polymer group may have a molecular weight of greater thanabout 300 Da, or alternatively greater than about 800 Da, or a molecularweight ranging from about 800 Da to about 3000 Da. The reactive groupmay form a covalent bond with an amino, a sulfhydryl or a hydroxynucleophile. For example, the reactive group may be an isothiocyanate,an isocyanate, a monochlorotriazine, a dichlorotriazine, ahalogen-substituted pyridine, a halogen-substituted diazine, aphosphoramidite, a maleimide, an aziridine, a sulfonyl halide, an acidhalide, a hydroxysuccinimidyl ester, a hydroxysulfosuccinimidyl ester, atetrafluorophenol ester, an imido ester, a hydrazine, anazidonitrophenyl, an azide, an alkyne, a 3-(2-pyridyldithio)-propionamide, a glyoxal or an aldehyde.

A fluorescence excitation wavelength of the compound of the inventionmay range from about 350 to about 1200 nm, while a fluorescence emissionwavelength of the compound may range from about 360 to 1250 nm.

In one embodiment, the compound of Formula I comprises a fluorophorewhich is a coumarin of the formula:

wherein one moiety of R_(a), R_(b), R_(c), R_(d), R_(e) and R_(f) is abond connecting said fluorophore to said moiety -(L)_(m)- or said moiety

also each remaining moiety of R_(a), R_(b), R_(c), R_(d), R_(e) andR_(f) has the formula (R)_(p)-(L)_(q)-, wherein

each R of R_(a), R_(b), R_(c), R_(d), R_(e) and R_(f) is independentlyi) a reactive group capable of forming a covalent bond upon reactingwith a reaction partner; ii) a radical of a water-soluble polymer; iii)an alkyl group, a trifluoroalkyl group, a halogen group, a sulfonylgroup, a sulfonate group, a phosphonate group or a sulfonamido group; oriv) —H;

each L of R_(a), R_(b), R_(c), R_(d), R_(e) and R_(f) is a a linkingmoiety formed of one or more chemical bond and containing about 1-100atoms selected such that the group is a stable moiety; each p of R_(a),R_(b), R_(c), R_(d), R_(e) and R_(f) is independently an integer rangingfrom 1 to 20; and each q of R_(a), R_(b), R_(c), R_(d), R_(e) and R_(f)is independently an integer ranging from 0 to 20.

In other embodiments, the compound of Formula I comprises a fluorophorewhich is a rhodamine of the formula:

wherein

connects said fluorophore to said moiety -(L)_(m)- or said moiety

R₄, R₅, and R₆ are each independently (R)_(p)-(L)_(q)-; each R of R₄,R₅, R₆ is independently i) a reactive group capable of forming acovalent bond upon reacting with a reaction partner; ii) a water solublepolymer group; iii) an alkyl group, a trifluoroalkyl group, a halogengroup, a sulfonate group or a sulfonamido group; or iv) —H; each L ofR₄, R₅ and R₆ is independently a linking moiety formed of one or morechemical bonds and containing about 1-100 atoms; each p of R₄, R₅, andR₆ is independently an integer ranging from 1 to 20; each q of R₄, R₅,and R₆ is independently an integer ranging from 0 to 20; and a, b, and care independently 0, 1, 2, or 3.

In related embodiments,

has the formula

In some embodiments, R₁, comprises a water soluble polymer group and R₂comprises a reactive group. For example, R₁, comprise a polyethyleneglycol. In other embodiments, R₂ comprises an N-hydroxysuccinimidegroup.

In still other embodiments, the fluorophore is substituted by one ormore sulfonate groups.

In another aspect, the invention provides a compound having a maximalfluorescence excitation wavelength, wherein the compound has a structureof Formula II:F—Y=Ψ  Formula II

Wherein F is a moiety having the structure:

Z⁻ is a counterion; Y is a bridge unit permitting electrondelocalization between F and Ψ;

Ψ is a moiety having one of the following structures:

X₁ and X₄ are independently

X₂ and X₃ are independently

a and b are independently 0, 1, 2, or 3;

b′ is 0, 1 or 2;

wherein when Ψ is Formula 1, and the maximal fluorescence excitationwavelength of the compound is less than 660 nm, then R₅ and R₆ areindependently (R)_(p) (L)_(q)-, wherein R₅ and R₆ are not combinable toform a substituted ring;

R₁, R₂, R₃, R₄, R₅, and R₆ are each independently (R)_(p)-(L)_(q)-,

each R of each (R)_(p)-(L)_(q)- of the compound is independently i) areactive group capable of forming a covalent bond upon reacting with areaction substrate; ii) a water soluble polymer group; iii) an alkylgroup, an aryl group, an alkylamino group, a dialkylamino group, analkoxy group, a trifluoroalkyl group, a halogen group, a sulfonyl group,a sulfonate group or a sulfonamido group; or iv) —H;

each L of each (R)_(p)-(L)_(q)- of the compound is independently alinking moiety formed of one or more chemical bonds and containing about1-100 atoms;

each p of each (R)_(p) (L)_(q)- is independently an integer of about 1to about 20;

each q of each (R)_(p)-(L)_(q)- of R₁ or R₂ is independently an integerof 0 to about 20;

each q of each (R)_(p)-(L)_(q)- of R₃, R₄, R₅, R₆, or R₇, isindependently an integer of 1 to about 20;

c is 0 or 1; d is 0 or 1;

at least one R of the (R)_(p)-(L)_(q)- of the compound is a reactivemoiety; and

at least one R of the (R)_(p)-(L)_(q)- of the compound is awater-soluble polymer.

In some embodiments, the invention provides the compound of Formula IIwherein when at least two adjacent R₁, and/or two adjacent R₂ arepresent, the two adjacent R₁, and/or the two adjacent R₂ are combinableto form a 6-membered ring which is unsubstituted or substituted by oneor more (R)_(p) (L)_(q)-. In some embodiments, the invention provides acompound of Formula II wherein when the two adjacent R₁, and/or the twoadjacent R₂ are combinable to form a 6-membered ring, the ring so formedis aromatic.

In some embodiments in the compounds of Formula II, when Ψ is Formula 1,and the maximal fluorescence excitation wavelength of the compound isequal to or greater than 660 nm, or Ψ is other than Formula 1, then R₅and R₆ are independently (R)_(p)-(L)_(q), or R₅ and R₆ are combinable toform a cyclic moiety which is unsubstituted or substituted by one ormore (R)_(p)-(L)_(q)-.

In some embodiments in the compounds of Formula II, Y is:

wherein when C is present, it is a five- or six-membered cyclic group;R₇ is (R)_(p)-(L)_(q)-; each R of (R)_(p)-(L)_(q)- is independently i) areactive group capable of forming a covalent bond upon reacting with areaction substrate; ii) a water soluble polymer group; iii) an alkylgroup, an aryl group, an alkylamino group, a dialkylamino group, analkoxy group, a trifluoroalkyl group, a halogen group, a sulfonyl group,a sulfonate group or a sulfonamido group; or iv) —H; each L of (R)_(p)(L)_(q)- is independently a linking moiety formed of one or morechemical bonds and containing about 1-100 atoms; p is an integer ofabout 1 to about 20; and q, is an integer of 1 to about 20.

In some embodiments of the compound of Formula II, at least one R of R₁and R₂ is a charged moiety. In other embodiments, at least one R of R₁and R₂ comprises a sulfonate group or a phosphonate group. In yet otherembodiments, each R of R₁ and R₂ comprises a sulfonate group or aphosphonate group.

In some embodiments of the compound of Formula II, X₂ and X₃ areindependently

In other embodiments, X₂ and X₃ are independently

In some embodiments of the compound of Formula II, the water-solublepolymer is a polyalkylene oxide. In other embodiments, the water-solublepolymer is a polyethylene oxide. In some embodiments, the water-solublepolymer has a molecular weight of greater than about 300. In otherembodiments, the water-soluble polymer has a molecular weight of greaterthan about 800. In other embodiments, the water-soluble polymer has amolecular weight ranging from about 800 to about 3000.

In some embodiments of the compound of Formula II, two adjacent (R₁)_(a)and the atoms in ring A to which it is attached are combined to form acarbocyclic ring. In some embodiments, the carbocyclic ring is aromatic.In other embodiments, two adjacent (R₂)_(b) and the atoms in ring B towhich it is attached are combined to form a carbocyclic ring. In someembodiments, the carbocyclic ring is aromatic.

In some embodiments of the compound of Formula II, the compound has theformula:

In some embodiments of the compound of Formula II, the compound has theformula:

wherein c is 1; d is 1; at least one R of R₃ and R₄ is a reactive groupcapable of forming a covalent bond upon reacting with a reactionsubstrate; and at least one R of R₃ and R₄ is a water soluble polymergroup.

In some embodiments of the compound of Formula II, the compound has theformula:

wherein c is 1; d is 1;

at least one R of R₃, R₄ and R₅ is a reactive group capable of forming acovalent bond upon reacting with a reaction substrate; and at least oneR of R₃, R₄ and R₅ is a radical of a water-soluble polymer.

In some embodiments of the compound of Formula II, the compound has theformula:

wherein c is 1; d is 1; one R of R₃ and R₄ is a reactive group capableof forming a covalent bond upon reacting with a reaction substrate; andone R of R₃ and R₄ is a water soluble polymer group.

In some embodiments of the compound of Formula II, the compound has theformula:

wherein c is 1; d is 1; at least one R of R₃, R₄ and R₅ is a reactivegroup capable of forming a covalent bond upon reacting with a reactionsubstrate; and at least one R of R₃, R₄ and R₅ is a radical of awater-soluble polymer.

In some embodiments of the compound of Formula II, the compound has theformula:

wherein c is 1; d is 1; at least one R of R₃, R₄ and R₅ is a reactivegroup capable of forming a covalent bond upon reacting with a reactionsubstrate; and at least one R of R₃, R₄ and R₅ is a radical of awater-soluble polymer.

In some embodiments, the maximal fluorescence excitation wavelength ofthe compound ranges from about 350 to about 1200 nm. In otherembodiments, a maximal fluorescence emission wavelength of the compoundranges from about 360 to about 1250 nm.

In some embodiments, the water-soluble polymer is a polyalkylene oxide.In other embodiments, the water-soluble polymer is a polyethylene oxide.In yet other embodiments, the water-soluble polymer is a carbohydrate.In other embodiments, the water-soluble polymer is a polypeptide. Insome embodiments, the water-soluble polymer has a molecular weight ofgreater than 300. In other embodiments, the water-soluble polymer has amolecular weight of greater than 800.

In some embodiments of the compound of Formula II, the compound is

In another aspect, the invention provides a substituted cyanine dyecomprising a reactive group and one or more water soluble polymergroups, wherein the cyanine dye has a maximal fluorescence excitationwavelength of equal to or greater than about 660 nm. In someembodiments, the substituted cyanine dye of claim, the dye issubstituted by a non-spiro moiety.

In a further aspect, the invention provides a substituted cyanine dyecomprising a reactive group and one or more water soluble polymergroups, wherein the cyanine dye has an absorption maximal wavelength ofequal to or greater than about 660 nm. In some embodiments, thesubstituted cyanine dye of claim, the dye is substituted by a non-spiromoiety.

In yet another aspect, the invention provides a binding agent labeledwith a substituted cyanine dye of the invention, wherein the bindingagent binds selectively to a target polypeptide to form a complex, andwherein formation of the complex yields a signal to noise ratio offluorescence that is at least 100. In some embodiments, the bindingagent comprises a substituted cyanine dye which is substituted with anon-spiro substituent. In some embodiments, the binding agent is apolypeptide. In other embodiments, the polypeptide is an antibody.

In another aspect, the invention provides a compound of Formula III:

wherein Z is —H, alkyl, —CF₃, —CN or a radical of the formula:

X₁ is ═O, ═NH₂ ⁺ or ═NR₆R₇ ⁺;

X₂ is —OH, NH₂, or —NR₈R₉;

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are each independently(R)_(p)-(L)_(q)-;

each R of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is independently i) areactive group capable of forming a covalent bond upon reacting with areaction substrate; ii) a water soluble polymer group; iii) an alkylgroup, a trifluoroalkyl group, a halogen group, a sulfonate group or asulfonamido group; or iv) —H;

each L of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is independently alinking moiety formed of one or more chemical bonds and containing about1-100 atoms;

each p of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is independently aninteger ranging from 1 to 20;

each q of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is independently aninteger ranging from 0 to 20;

a, b, c, d and e are independently 0, 1, 2, 3 or 4;

at least one R of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a reactivemoiety; and

at least one R of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a watersoluble polymer group.

In some embodiments of the compound of Formula III, at least one of R₆,R₇, R₈ or R₉ is combinable with a neighboring R₁ or R₂ and anyintervening atoms in a ring to which the neighboring R₁ or R₂ isattached, to form a 5- or 6-membered ring which is unsubstituted orsubstituted by one or more (R)_(p)-(L)_(q)-. In some embodiments, whenthe at least one of R₆, R₇, R₈ or R₉ and a neighboring R₁ or R₂ arecombinable to form a 5- or 6-membered ring, the ring so formed isunsaturated. In other embodiments, when the at least one of R₆, R₇, R₈or R₉ and a neighboring R₁ or R₂ are combinable to form a 5- or6-membered ring, the ring so formed is saturated.

In some embodiments of the compound of Formula III, X₁ is ═NH₂ ⁺. Inother embodiments, X₁ is ═O. In yet other embodiments, X₂ is —OH. Insome embodiments X₂ is —NH₂.

In some embodiments of the compound of Formula III, a maximalfluorescence excitation wavelength of the compound ranges from about 450to 750 nm. In other embodiments, a maximal fluorescence emissionwavelength of the compound ranges from about 470 to 800 nm.

In another aspect, the invention provides a compound of Formula V:

wherein R₁, R₂, R₃, and R₄ are each independently (R)_(p)-(L)_(q)-,

each R of R₁, R₂, R₃, and R₄ is independently i) a reactive groupcapable of forming a covalent bond upon reacting with a reactionsubstrate; ii) a water soluble polymer group; iii) an alkyl group, atrifluoroalkyl group, a halogen group, a phosphonate group, a sulfonategroup or a sulfonamido group; or iv) —H;

each L of R₁, R₂, R₃, and R₄ is independently a linking moiety formed ofone or more chemical bond and containing about 1-100 atoms;

each p of R₁, R₂, R₃, R₄, R₅, R₆ and R₇ is independently an integerranging from 1 to 20;

each q of R₁, R₂, R₃, R₄, R₅, R₆ and R₇ is independently an integerranging from 0 to 20;

a, b, c and d are independently 0, 1, 2 or 3;

at least one R of R₁, R₂, R₃ and R₄ is a reactive moiety; and

at least one R of R₁, R₂, R₃ and R₄ is a water soluble polymer group.

In a further aspect, the invention provides a kit comprising: i) thecompound of claim 1, 25, 59, 60, 66, or 76; ii) a buffer; iii) materialsor devices for purifying conjugation products; and iv) instructionsinstructing the use of the compound.

In another aspect, the invention provides a biomolecule comprising alabel having a structure of Formula I, II, III, IV, V, a substitutedcyanine dye comprising a reactive group and one or more water solublepolymer groups, wherein the cyanine dye has a maximal fluorescenceexcitation wavelength of equal to or greater than about 660 nm or asubstituted cyanine dye comprising a reactive group and one or morewater soluble polymer groups, wherein the cyanine dye has an absorptionmaximal wavelength of equal to or greater than about 660 nm, wherein theat least one reactive moiety of the Formula has undergone a reactionwhich attaches the label to the biomolecule. In some embodiments, thebiomolecule comprises a polynucleotide. In some embodiments, thebiomolecule comprises a polypeptide. In some embodiments, thepolypeptide further comprises an antigen binding site. In someembodiments, the polypeptide is a whole immunoglobulin. In someembodiments, the polypeptide is a Fab fragment.

In another aspect, the invention provides an immunoglobin comprising alabel having a structure of Formula I, II, III, IV, V, a substitutedcyanine dye comprising a reactive group and one or more water solublepolymer groups, wherein the cyanine dye has a maximal fluorescenceexcitation wavelength of equal to or greater than about 660 nm or asubstituted cyanine dye comprising a reactive group and one or morewater soluble polymer groups, wherein the cyanine dye has an absorptionmaximal wavelength of equal to or greater than about 660 nm, wherein theat least one reactive moiety of the Formula has undergone a reactionwhich attaches the label to the immunoglobin, wherein the immunoglobinis an antibody that binds specifically to an antigen on a cancer cell.In some embodiments, the antibody binds to erb2.

In another aspect, the invention provides a method of preparing alabeled biomolecule comprising reacting a compound having a structure ofFormula I, II, III, IV, V, a substituted cyanine dye comprising areactive group and one or more water soluble polymer groups, wherein thecyanine dye has a maximal fluorescence excitation wavelength of equal toor greater than about 660 nm or a substituted cyanine dye comprising areactive group and one or more water soluble polymer groups, wherein thecyanine dye has an absorption maximal wavelength of equal to or greaterthan about 660 nm, and a substrate biomolecule under conditionssufficient to effect crosslinking between the compound and the substratebiomolecule. In some embodiments, the substrate biomolecule is apolypeptide, a polynucleotide, a carbohydrate, a lipid or a combinationthereof. In other embodiments, the substrate biomolecule is apolynucleotide.

In yet another aspect, the invention provides a method for labeling acell within a population of cells whereby the cell is differentiallylabeled relative to neighboring cells within the population, the methodcomprising contacting the cell with a biomolecule of labeled accordingto the methods of the invention, wherein the biomolecule comprises atargeting moiety that binds to a binding partner that is indicative ofthe cell, and thereby differentially labeling the cell relative toneighboring cells within the population. In some embodiments, the methodfurther comprises the step of imaging the cell, the imaging stepcomprising: i) directing exciting wavelength to the cell; and ii)detecting emitted fluorescence from the cell. In some embodiments, thelabeling takes place in vitro. In other embodiments, the labeling takesplace in vivo.

In another aspect, the invention provides an immunoglobulin labeled witha fluorescent compound comprising a polyalkylene oxide and a fluorophorethat has an absorption maximal wavelength equal to or greater than 685nm. In some embodiments, the immunoglobulin retains binding specificityto a target upon conjugation to the fluorescent compound. In someembodiments, the immunoglobin is an antibody that binds specifically toan antigen on a cancer cell. In some embodiments, the antibody binds toerb2. In some embodiments, the fluorescent compound is a compound ofFormula I, II, III, a substituted cyanine dye comprising a reactivegroup and one or more water soluble polymer groups, wherein the cyaninedye has a maximal fluorescence excitation wavelength of equal to orgreater than about 660 nm or a substituted cyanine dye comprising areactive group and one or more water soluble polymer groups, wherein thecyanine dye has an absorption maximal wavelength of equal to or greaterthan about 660 nm.

In another aspect, the invention provides an immunoglobulin labeled witha fluorescent compound comprising a polyalkylene oxide and a fluorophorethat has an absorption maximal wavelength at or greater than 750 nm. Insome embodiments, the immunoglobulin retains binding specificity to atarget upon conjugation to the fluorescent compound. In someembodiments, the immunoglobin is an antibody that binds specifically toan antigen on a cancer cell. In some embodiments, the antibody binds toerb2. In some embodiments, the fluorescent compound is a compound havinga structure of Formula I, II, III, V, a substituted cyanine dyecomprising a reactive group and one or more water soluble polymergroups, wherein the cyanine dye has a maximal fluorescence excitationwavelength of equal to or greater than about 660 nm or a substitutedcyanine dye comprising a reactive group and one or more water solublepolymer groups, wherein the cyanine dye has an absorption maximalwavelength of equal to or greater than about 660 nm.

In yet a further aspect, the invention provides a polypeptide labeledwith a fluorescent compound, the polypeptide exhibiting a serumhalf-life no shorter than that of a corresponding polypeptide that lacksthe fluorescent compound, wherein the fluorescent compound is a compoundhaving a structure of Formula I, II, III, V, a substituted cyanine dyecomprising a reactive group and one or more water soluble polymergroups, wherein the cyanine dye has a maximal fluorescence excitationwavelength of equal to or greater than about 660 nm or a substitutedcyanine dye comprising a reactive group and one or more water solublepolymer groups, wherein the cyanine dye has an absorption maximalwavelength of equal to or greater than about 660 nm.

In another aspect, the invention provides a method of labeling apolypeptide comprising: forming a complex that comprises the polypeptideand a binding agent, wherein the binding agent comprises a fluorescentlabel having a structure of Formula I, II, III, V, a substituted cyaninedye comprising a reactive group and one or more water soluble polymergroups, wherein the cyanine dye has a maximal fluorescence excitationwavelength of equal to or greater than about 660 nm or a substitutedcyanine dye comprising a reactive group and one or more water solublepolymer groups, wherein the cyanine dye has an absorption maximalwavelength of equal to or greater than about 660 nm, wherein the atleast one reactive moiety of the Formula has undergone a reaction whichattaches the label to the binding agent. In some embodiments, thebinding agent is an antibody. In some embodiments, the complex comprises(a) a primary antibody that binds to the polypeptide, and (b) thebinding agent which functions as a secondary antibody exhibiting bindingcapability to the primary antibody. In some embodiments, the labelingoccurs on a solid substrate. In some embodiments, that labels apolypeptide intracellularly. In some embodiments, the complex yields asignal to noise ratio greater than about 100, wherein the signal tonoise ratio is calculated by the formula: (fluorescent signal from acomplex comprising the polypeptide bound by a primary antibody which inturn is bound to the binding agent)/(fluorescent signal from a mixtureof the polypeptide, an isotype control primary antibody and the bindingagent). In other embodiments, the complex yields a signal to noise ratiogreater than about 250, wherein the signal to noise ratio is calculatedby the formula: (fluorescent signal from a complex comprising thepolypeptide bound by a primary antibody which in turn is bound to thebinding agent)/(fluorescent signal from a mixture of the polypeptide, anisotype control primary antibody and the binding agent). In yet otherembodiments, the complex yields a signal to noise ratio greater thanabout 270, wherein the signal to noise ratio is calculated by theformula: (fluorescent signal from a complex comprising the polypeptidebound by a primary antibody which in turn is bound to the bindingagent)/(fluorescent signal from a mixture of the polypeptide, an isotypecontrol primary antibody and the binding agent).

In some embodiments, the complex yields a total fluorescence signal atleast 5% greater than that generated by a complex formed with the sameprimary antibody and with the same secondary antibody that has acomparable degree of labeling with a DyLight 680™ dye. In otherembodiments, the complex yields a total fluorescence signal at least 5%greater than that generated by a complex formed with the same primaryantibody and with the same secondary antibody that has a comparabledegree of labeling with a Cy5.5® dye. In yet other embodiments, thecomplex yields a total fluorescence signal at least 5% greater than thatgenerated by a complex formed with the same primary antibody and withthe same secondary antibody that has a comparable degree of labelingwith an Alexa Fluor 680® dye. In some embodiments, the label having astructure of a Formula of Formula II is the compound having thestructure:

In some embodiments, each complex is excited at 635 nm or 633 nm. Insome embodiments, each complex is present at identical proteinconcentrations.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a graphical representation showing the absorption spectra ofCy5® dye and Compound No. 41 (Example 41), respectively, conjugated togoat anti-mouse IgG at similar degrees of labeling (i.e., ˜4) in anaqueous buffer. The spectrum of Cy5® dye displays a double peakcharacteristic of dye aggregation while the spectrum of Compound No. 41shows mainly a single peak, indicating a substantial lack of dyeaggregation.

FIG. 2 is a graphical representation showing the fluorescence emissionspectra of goat anti-rabbit IgG conjugates of Cy5® dye and Compound No.41 (Example 41) at similar degrees of labeling (i.e., ˜5) and equivalentprotein concentrations, when excited at 630 nm. The data demonstratesthat the fluorescent group of the invention is significantly morefluorescent than Cy5® dye.

FIG. 3 is a plot of total fluorescence vs. degree of labeling (DOL) forgoat anti-rabbit IgG conjugates of Compound No. 41 (Example 41) and Cy5®dye at identical protein concentrations in an aqueous buffer, whenexcited at 630 nm. The data shows that the fluorescent group of theinvention has less fluorescence quenching than Cy5® dye when theantibody is at higher degrees of labeling. (See Example 97.)

FIG. 4 is a plot of total fluorescence vs. degree of labeling (DOL) forstreptavidin conjugates of Compound No. 5 (Example 5), Cy3® dye andAlexa Fluor 555® dye at identical protein concentrations in an aqueousbuffer, when excited at 530 nm. The higher slope given by thefluorescent group of the invention demonstrates that the fluorescentgroup of the invention is intrinsically more fluorescent than eitherCy3® dye or Alexa Fluor 555® dye. (See Example 97.)

FIG. 5 is a plot of total fluorescence vs. degree of labeling (DOL) forgoat anti-mouse conjugates of Compound No. 5 (Example 5), Cy3® dye andAlexa Fluor 555® dye at identical protein concentrations in an aqueousbuffer, when excited at 530 nm. All three fluorescent groups havesimilar absorption and emission maxima, but Cy3® dye and Alexa Fluor555® dye do not have a water soluble polymer group. The data shows thatthe fluorescent group of the invention is as bright as Alexa Fluor 555®dye over a wide range of degree of labeling. However, the fluorescenceof Cy3® dye is substantially quenched at higher degrees of labeling.(See Example 97.)

FIG. 6 is a plot of total fluorescence vs. degree of labeling (DOL) forgoat anti-mouse IgG conjugates of Compound No. 67 (Example 67) and aspectrally similar fluorescent group without a water soluble polymergroup, Alexa Fluor 660® dye, at identical protein concentrations in anaqueous buffer, when excited at 640 nm. The data shows that, compared toAlexa Fluor 660® dye, the fluorescent group of the invention has higherfluorescence quantum yield over a wide of degree of labeling and hasless fluorescence quenching when the antibody is at higher degrees oflabeling. (See Example 97.)

FIG. 7 is a plot of total fluorescence vs. degree of labeling (DOL) forgoat anti-mouse IgG conjugates of Compound No. 92 (Example 92) and asimilar fluorescent group without a water soluble polymer group, AMCA,at identical protein concentrations in an aqueous buffer, when excitedat 350 nm. The data shows that, compared to AMCA, the fluorescent groupof the invention has higher fluorescence quantum yield over a wide ofdegree of labeling and has less fluorescence quenching when the antibodyis at higher degrees of labeling. (See Example 97.)

FIG. 8 is a plot of total fluorescence vs. degree of labeling (DOL) forgoat anti-mouse IgG conjugates of Compound No. 96 (Example 96) and asimilar fluorescent group without a water soluble polymer group,5(6)-carboxyrhodamine 110 (CR110), at identical protein concentrationsin an aqueous buffer, when excited at 488 nm. The data shows that,compared to CR110, the fluorescent group of the invention has higherfluorescence quantum yield over a wide of degree of labeling and hasless fluorescence quenching when the antibody is at higher degrees oflabeling. (See Example 97.)

FIG. 9 is a graphical representation showing the relative fluorescencelevels of Jurkat cells stained with various fluorescently labeledantibodies as measured by flow cytometry. The cells were first labeledwith mouse anti-human CD3 antibody and then stained with goat anti-mouseIgG labeled with Compound No. 41 at a degree of labeling of 2.7, 3.9 and5.8, respectively, or with a commercially available goat anti-mouse IgGlabeled with Cy5® dye ˜(dark columns). To measure the backgroundfluorescence from each labeled secondary antibody, the cells were alsostained directly with each of the fluorescent secondary antibody withoutthe primary antibody (blank columns). The results show that cellsstained with antibody conjugates of this invention are 25-60% brighterthan cells stained with the commercial antibody conjugate, withexcellent signal to noise ratio. (See Example 104.)

FIG. 10 is a flow cytometry histogram showing the distribution ofimmunofluorescently stained cells as a function of fluorescenceintensity. Jurkat cells were fixed, permeabilized, and incubated withmouse anti-human CD3 antibody. The CD3 antibody was followed byincubation with goat anti-mouse IgG labeled with AlexaFluor647® dye (DOL3.1) (gray-lined peak) or with compound No. 41 (DOL 3.9) (dark-linedpeak). The figure shows that AlexaFluor647® dye-labeled antibody gavemore weakly stained cells and more scattered fluorescence staining thanthe antibody labeled with the fluorescent group of the invention. (SeeExample 105.)

FIG. 11 shows a comparison of the photostability of a fluorescent groupof the invention over time compared to the fluorescein and Alexa Fluor488® dye. Actin filaments were stained with phalloidin labeled withcompound No. 96, Alexa Fluor 488® dye or fluorescein and the relativefluorescence of each sample was plotted vs. time. FIG. 11 shows that thephotostability of a biomolecule labeled using compound 96 is superior tofluorescein as well as Alexa Fluor 488® dye. (See Example 107.)

FIGS. 12A, B, and C are graphical representations showing the absorptionspectra of goat anti-mouse IgG conjugates prepared at different degreeof labeling (DOL) with three near-IR dyes: FIG. 12A represents theabsorption spectra of the conjugate formed from labeling goat anti-mouseIgG with Cy7® dye at 4 different DOL (1.2 to 3.4 dye molecules perantibody). FIG. 12B represents the absoprtion specta of the conjugateformed from labeling goat anti-mouse IgG with Alexa Fluor 750® (AF750®)dye at four different DOL (2.2 to 5.9 dye molecules per antibody). FIG.12C represents the absorption spectra of the conjugate formed fromlabeling goat anti-mouse IgG with Dye No. 29 (Table 3), at six differentDOL (1.1 to 7.4 dye molecules per antibody). All spectra were taken atroom temperature in PBS 7.4 buffer. The spectra of both Cy7® dye- andAF750® dye-labeled conjugates (A and B) display a double peakcharacteristic of dye aggregation while the spectra of Compound No.29-labeled conjugates (C) show mainly a single peak, indicating asubstantial lack of dye aggregation.

FIG. 13 is a graphical representation of total fluorescence vs. degreeof labeling (DOL) for goat anti-mouse IgG conjugates labeled with anear-IR dye of the invention, Dye No. 29 (Table 3), and goat anti-mouseIgG conjugates labeled each labeled with one of two other spectrallysimilar near-IR fluorescent groups without a water soluble polymergroup, Alexa Fluor 750® (AF750®) dye and Cy7® dye, at identical proteinconcentrations in pH 7.4 PBS buffer, when excited at 735 nm. The datashows that, compared to AF750® dye and Cy7® dye, the fluorescent groupof Dye 29 has higher fluorescence quantum yield over a wide degree oflabeling and has less fluorescence quenching when the antibody is athigher degrees of labeling.

FIG. 14 is a graphical representation showing the relative fluorescencelevels of Jurkat cells stained with goat anti-mouse IgG labeled with anear-IR dye of the invention, Dye No. 29 (Table 3), Alexa Fluor 750® dyeand Cy7® dye, respectively, as measured by flow cytometry. The cellswere first labeled with mouse anti-human CD3 antibody and then stainedwith one of the three labeled secondary antibodies, which all have asimilar degree of labeling. To measure the background fluorescence fromeach labeled secondary antibody, the cells were also stained with anisotype control primary antibody and each of the fluorescent secondaryantibody (dark columns). The results show that cells stained withantibody conjugate of this invention are significantly brighter and haveexcellent signal-to-noise ratio.

FIG. 15 compares the photostability of three near-IR dyes: Compound No.29 of Table 3, Alexa Fluor 750® (AF750®) dye and Cy7® dye. Solutions ofthe three dyes at 5 μM dye concentration were exposed to sun light for ½hour. Absorption spectra of the solutions were recorded before and afterthe photolysis. The results show the near-IR dye of the invention issignificantly more stable than both AF750® dye and Cy7® dye.

FIGS. 16A and B are graphical representations for relative fluorescencelevels of Jurkat cells stained intracellularly with an antibody labeledwith Alexa Fluor 750® (AF750®) dye or Dye 29 (Table 3). FIG. 16A is agraphical representation showing the relative fluorescence levels ofJurkat cells stained with indicated amount of either goat anti-mouse IgGlabeled with compound No. 29 (DOL=3.5) or a commercially available goatanti-mouse IgG labeled with an APC-AF750® tandem dye (Invitrogen), asmeasured by flow cytometry. The cells were first labeled with mouseanti-human CD3 antibody and then stained with one of the two labeledsecondary antibodies. To measure the background fluorescence from eachlabeled secondary antibody, the cells were also stained with an isotypecontrol primary antibody and each of the fluorescent secondary antibody(darkened columns). FIG. 16B is a graphical representation showing thesignal-to-noise ratios of the stainings from FIG. 16A. Flow cytometryexperiments were performed on a BD LSR II equipped with a 633 nm laserand 780/60 nm PMT detector. The results show that Dye No. 29 gives highfluorescent signal with very little background while the APC-AF750®tandem dye showed significant nonspecific staining. The results showthat Dye No. 29 has very little absorption at 633 nm whereas the tandemdye has nearly maximal absorption at the laser wavelength due to thedonor dye APC. Dye No. 29, near-IR dyes the like disclosed herein areparticularly advantageous for flow cytometry analysis using a 633 nm orlonger wavelength excitation source.

FIG. 17 is a graphical representation of total fluorescence vs. degreeof labeling (DOL) for goat anti-mouse IgG conjugates of three near-IRdyes with similar wavelengths: Dye No. 31 (Table 3), Dylight™ 800Dyefrom Thermo Fisher and IRDye 800® CW dye from Li-Cor Biosciences. Thedata show that Dye No. 31 is significantly brighter than the other twodyes over a wide range of DOL.

FIG. 18 is a graphical representation of the relative fluorescencelevels of Jurkat cells stained with goat anti-mouse IgG antibodieslabeled with Dye No. 31 (Table 3), Dylight™ 800 from Thermo Fisher andIRDye 800®CW dye from Li-Cor Biosciences, respectively, as measured byflow cytometry. To assess the fluorescence quenching of the threenear-IR dyes, the antibody was labeled with each dye at different degreeof labeling (DOL) as indicated. The cells were first labeled with mouseanti-human CD3 antibody and then stained with one of the labeledsecondary antibodies. To measure the background fluorescence from eachlabeled secondary antibody, the cells were also stained with an isotypecontrol primary antibody and each of the fluorescent secondaryantibodies (isotype, dark columns). The results show that Dye No. 31 issignificantly brighter than both Dylight™ 800 dye and IRDye 800® CW dyeover a wide range of DOL. Also importantly, Dye No. 31 produced muchbetter signal-to-noise ratio than the other two dyes.

FIG. 19 is a plot of total fluorescence vs. degree of labeling (DOL) forgoat anti-mouse IgG conjugates of a near-IR dye of the invention, DyeNo. 32 (Table 3), and three spectrally similar near-IR fluorescentgroups, Cy5.5® dye from GE Healthcare, Alexa Fluor 680® (AF680) dye fromInvitrogen and Dylight™ 680 from Thermo Fisher, respectively.Fluorescence measurements were made in pH 7.4 PBS buffer using 660 nmexcitation. The data shows that, compared to Cy5.5®, Alexa Fluor 680®and Dylight™ 680, the fluorescent group of the invention has higherfluorescence quantum yield over a wide degree of labeling.

FIGS. 20A and B are graphical representations of data related to therelative fluorescence level of Jurkat cells stained with goat anti-mouseIgG antibodies labeled individually with four different near IR dyes.FIG. 20A is a graphical representation showing the relative fluorescencelevels of Jurkat cells stained with goat anti-mouse IgG antibodieslabeled with Dye No. 32 (Table 3), Cy5.5® dye from GE Healthcare,Dylight™ 680 dye from Thermo Fisher or Alexa Fluor 680® (AF680®) dyefrom Invitrogen, as measured by flow cytometry. To assess thefluorescence quenching of the three near-IR dyes, each portion of goatanti-mouse IgG antibody was labeled with one dye at one of two differentdegree of labeling (DOL) as indicated to form a set of eight antibodies.The cells were first labeled with mouse anti-human CD3 antibody and thenstained with one of the labeled secondary antibodies. To measure thebackground fluorescence from each labeled secondary antibody, the cellswere also stained with an isotype control primary antibody and each ofthe fluorescent secondary antibodies (isotype, dark columns). FIG. 20Bis a plot of signal-to-noise ratio (S/N) for the staining results inFIG. 20A. The data show that conjugates labeled with Dye No. 32 are muchbrighter and more specific in staining than conjugates prepared from theother three commercial near-IR dyes.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The present invention discloses fluorescent compounds comprising atleast one reactive group and at least one water soluble polymer. Suchcompounds may have desirable properties such as restrictedintramolecular mobility, increased fluorescence quantum yield, decreasedaggregation, increased solubility, decreased quenching and increased invivo and in vitro stability. The compounds may be used for labelingmolecules and biomolecules such as polypeptides and polynucleotides andare suitable for use in a wide range of applications, includingdiagnostic and imaging systems.

Fluorescent compounds and labeled molecules of the invention may exhibitreduced aggregation. Dye aggregation is often seen as a majorcontributing factor to fluorescence quenching. Prevention of aggregationin the present invention may be achieved without the use of an excessivenumber of negatively charged sulfonate groups. This in turn may aid inthe labeling of biomolecules such as proteins because the labeledprotein may have an isoelectric point comparable to that of thesubstrate protein, and may thereby better maintain its biologicalspecificity. The use of water soluble polymers of the invention may alsoaid in camouflaging or shielding the fluorophore to which it is linked.For example, such a water soluble polymer may be a relatively largegroup such as a polyethylene glycol moiety. This may be particularlydesirable when the fluorophore is linked to a biomolecule such as apolypeptide. Consequently, compared to other fluorescently labeledproteins, proteins labeled with the compounds of the present inventionmay be more resistant to protease-catalyzed degradation. Accordingly,labeled proteins, such as labeled antibodies, of the invention may havea longer half-life in the circulation when applied to an animal's body,such as a mammal's body, and therefore may be suitable for in vivoimaging. Labeled biomolecules may also exhibit a longer half-life in invitro systems, for example in assays employing serum or other biologicalextracts.

A water soluble polymer may restrict or reduce the intramolecularmobility, such as the vibration and rotation, of the fluorophore towhich it is attached. This may increase the fluorescence quantum yieldof the fluorescent group. The fluorescence enhancement effect may beparticularly effective for fluorescent groups that have a relativelyflexible core structure. Additionally, labeled molecules of theinvention may be less immunogenic and less antigenic in vivo than thecorresponding substrate biomolecules. Furthermore, the compounds andlabeled molecules of the invention may exhibit higher photostability andresistance to bleaching of the fluorescent group.

DEFINITIONS

The compounds of the present invention may have asymmetric centers,chiral axes, and chiral planes (as described in: E. L. Eliel and S. H.Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, NewYork, 1994, pages 1119 1190), and occur as racemates, racemic mixtures,and as individual diastereomers, with all possible isomers and mixturesthereof, including optical isomers, being included in the presentinvention. In addition, the compounds disclosed herein may exist astautomers and both tautomeric forms are intended to be encompassed bythe scope of the invention, even though only one tautomeric structure isdepicted.

When any variable (e.g. R, L, (R₁)_(a), (L)_(q)) occurs more than onetime in any constituent, its definition on each occurrence isindependent at every other occurrence. Combinations of substituents andvariables are permissible only if such combinations result in stablecompounds. Lines drawn into the ring systems from substituents indicatethat the indicated bond may be attached to any of the substitutable ringcarbon atoms. If the ring system is polycyclic, it is intended that thebond be attached to any of the suitable carbon atoms on the proximalring only. Substitution of a ring by a substitutent generally allows thesubstituent to be a cyclic structure fused to the ring.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.The phrase “optionally substituted with one or more substituents” shouldbe taken to be equivalent to the phrase “optionally substituted with atleast one substituent” and in such cases the preferred embodiment willhave from zero to three substituents.

As used herein, “alkyl” is intended to include both branched,straight-chain, and cyclic saturated aliphatic hydrocarbon groups. Alkylgroups specifically include methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, octyl, nonyl, decyl, and so on, as well as cycloalkyls such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, tetrahydronaphthalene,methylenecylohexyl, and so on. “Alkoxy” represents an alkyl groupattached through an oxygen bridge.

The term “alkenyl” refers to a non-aromatic hydrocarbon group, straight,branched or cyclic, containing at least one carbon to carbon doublebond. Alkenyl groups include, but are not limited to, ethenyl, propenyl,butenyl and cyclohexenyl. The straight, branched or cyclic portion ofthe alkenyl group may contain double bonds and may be substituted if asubstituted alkenyl group is indicated.

The term “alkynyl” refers to a hydrocarbon group, straight, branched orcyclic, containing at least one carbon to carbon triple bond. Alkynylgroups include, but are not limited to, ethynyl, propynyl and butynyl.The straight, branched or cyclic portion of the alkynyl group maycontain triple bonds and may be substituted if a substituted alkynylgroup is indicated.

As used herein, “aryl” is intended to mean any stable monocyclic orpolycyclic carbon ring of up to 7 atoms in each ring, wherein at leastone ring is aromatic. Examples of such aryl elements include phenyl,naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl oracenaphthyl. In cases where the aryl substituent is bicyclic and onering is non-aromatic, it is understood that attachment is via thearomatic ring.

The term “heteroaryl”, as used herein, represents a stable monocyclic orbicyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and contains from 1 to 4 heteroatoms selected from the groupconsisting of O, N and S. Heteroaryl groups within the scope of thisdefinition include but are not limited to acridinyl, carbazolyl,cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl,thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl,pyrrolyl, tetrahydroquinoline, xanthenyl, and coumarinyl. In cases wherethe heteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively.

The term “heterocycle” or “heterocyclyl” as used herein is intended tomean a 5- to 10-membered aromatic or nonaromatic heterocycle containingat least one heteroatom which is O, N or S. This definition includesbicyclic groups. “Heterocyclyl” therefore includes the above mentionedheteroaryls, as well as dihydro and tetrahydro analogs thereof. Furtherexamples of “heterocyclyl” include, but are not limited to thefollowing: benzoimidazolyl, benzofuranyl, benzofurazanyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl,indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl,pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl,thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl,hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl,thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl.

The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl andheterocyclyl substituents may be unsubstituted or unsubstituted, unlessspecifically defined otherwise. For example, an alkyl group may besubstituted with one or more substituents selected from OH, oxo, halo,alkoxy, dialkylamino, or heterocyclyl, such as morpholinyl orpiperidinyl.

The terms “halo” or “halogen” are intended to include chloro, fluoro,bromo and iodo groups.

The term “aromatic” is used in its usual sense, including unsaturationthat is essentially delocalized across multiple bonds, such as around aring.

The term “substituent” refers to an atom, radical or chemical groupwhich replaces a hydrogen in a substituted chemical group, radical,molecule, moiety or compound.

“Spiro” as used herein, refers to a cyclic moiety which is attached toanother group such that one of the ring atoms of the cyclic moiety isalso an atom of said other group. A non-spiro substituent is a moietycyclic or noncyclic which is directly attached to said other group viabond connection between atoms of the non-spiro moiety and said othergroup. An example of a spiro moiety is, for instance, a substitutentRing B on cyclohexanone Ring A.

Unless otherwise stated, the term “radical”, as applied to any moleculeor compound, is used to refer to a part, fragment or group of themolecule or compound rather than to a “free radical”. A radical may belinked to another moiety through a covalent bond.

The terms “polynucleotides”, “nucleic acids”, “nucleotides”, “probes”and “oligonucleotides” are used interchangeably. They refer to apolymeric form of nucleotides of any length, either deoxyribonucleotidesor ribonucleotides, or analogs thereof. Polynucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The following are non-limiting examples of polynucleotides:coding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.“Polynucleotide” may also be used to refer to peptide nucleic acids(PNA), locked nucleic acids (LNA), threofuranosyl nucleic acids (TNA)and other unnatural nucleic acids or nucleic acid mimics. Other base andbackbone modifications known in the art are encompassed in thisdefinition. See, e.g. De Mesmaeker et al (1997) Pure & Appl. Chem., 69,3, pp 437-440.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear, cyclic, or branched, it may comprisemodified amino acids, and it may be interrupted by non-amino acids. Theterms also encompass amino acid polymers that have been modified, forexample, via sulfonation, glycosylation, lipidation, acetylation,phosphorylation, iodination, methylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, ubiquitination, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” refers to either natural and/or unnatural or synthetic aminoacids, including glycine and both the D or L optical isomers, and aminoacid analogs and peptidomimetics.

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen-binding site which specifically binds(“immunoreacts with”) an antigen. Structurally, the simplest naturallyoccurring antibody (e.g., IgG) comprises four polypeptide chains, twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds. The immunoglobulins represent a large family of molecules thatinclude several types of molecules, such as IgD, IgG, IgA, IgM and IgE.The term “immunoglobulin molecule” includes, for example, hybridantibodies, or altered antibodies, and fragments thereof. It has beenshown that the antigen binding function of an antibody can be performedby fragments of a naturally-occurring antibody. These fragments arecollectively termed “antigen-binding units”. Antigen binding units canbe broadly divided into “single-chain” (“Sc”) and “non-single-chain”(“Nsc”) types based on their molecular structures.

Also encompassed within the terms “antibodies” are immunoglobulinmolecules of a variety of species origins including invertebrates andvertebrates. The term “human” as applies to an antibody or an antigenbinding unit refers to an immunoglobulin molecule expressed by a humangene or fragment thereof. The term “humanized” as applies to a non-human(e.g. rodent or primate) antibodies are hybrid immunoglobulins,immunoglobulin chains or fragments thereof which contain minimalsequence derived from non-human immunoglobulin. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, rabbit or primate having thedesired specificity, affinity and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, the humanized antibodymay comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. These modifications aremade to further refine and optimize antibody performance and minimizeimmunogenicity when introduced into a human body. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody may also comprise atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin.

The term “stable” refers to compositions and compounds which havesufficient chemical stability to survive isolation from a reactionmixture to a useful degree of purity for use in a desired application.

The terms “fluorescent group”, “fluorophore”, “dye” or “fluorescentgroup” refer interchangeably to molecules, groups or radicals which arefluorescent. The term “fluorescent” as applied to a molecule of compoundis used to refer to the property of the compound of absorbing energy(such as UV, visible or IR radiation) and re-emitting at least afraction of that energy as light over time. Fluorescent groups,compounds or fluorophores include, but are not limited to discretecompounds, molecules, proteins and macromolecular complexes.Fluorophores also include compounds that exhibit long-lived fluorescencedecay such as lanthanide ions and lanthanide complexes with organicligand sensitizers.

A “subject” as used herein refers to a biological entity containingexpressed genetic materials. The biological entity is in variousembodiments, a vertebrate. In some embodiment, the biological entity isa mammal. In other embodiments, the subject is a biological entity whichcomprises a human.

A “control” is an alternative subject or sample used in an experimentfor comparison purposes. A control can be “positive” or “negative”. Forexample, where the purpose of the experiment is to detect adifferentially expressed transcript or polypeptide in cell or tissueaffected by a disease of concern, it is generally preferable to use apositive control (a subject or a sample from a subject, exhibiting suchdifferential expression and syndromes characteristic of that disease),and a negative control (a subject or a sample from a subject lacking thedifferential expression and clinical syndrome of that disease.

The term “FRET” refers to Foerster resonance energy transfer. In thepresent invention, FRET refers to energy transfer processes occurringbetween at least two fluorescent compounds, between a fluorescentcompound and a non-fluorescent component or between a fluorescentcomponent and a non-fluorescent component.

A “binding agent” is a molecule that exhibits binding selectivitytowards a binding partner or a target molecule to which it binds. Abinding agent may be a biomolecule such as a polypeptide such as anantibody or protein, polypeptide-based toxin, amino acid, nucleotide,polynucleotides including DNA and RNA, lipids, and carbohydrates, or acombination thereof. A binding agent may also be a hapten, drug,ion-complexing agent such as metal chelators, microparticles, syntheticor natural polymers, cells, viruses, or other fluorescent moleculesincluding the dye molecule according to the invention.

A “targeting moiety” is the portion of the binding agent that binds to abinding partner. A targeting moiety may be, without limitation, anucleotide sequence within a polynucleotide that selectively binds toanother polynucleotide or polypeptide. Another nonlimiting example of atargeting moiety may be a polypeptide sequence within a largerpolypeptide sequence which binds specifically to a polynucleotidesequence or a second polypeptide sequence. A targeting moiety may be asmall molecule or structural motif which will bind to a proteinreceptor, another small molecule motif, or complexing agent, withoutlimitation. The selective binding may be a specific binding event.

A “binding partner” is a molecule or particle which is bound by thetargeting moiety. It can be a cell, virus, fragment of a cell, antibody,fragment of an antibody, peptide, protein, polynucleotide, antigen,small molecule, or a combination thereof. It may be bound selectively orspecifically by the binding agent.

The term “signal to noise ratio” of fluorescence as referred to hereinin the context of a polypeptide-antibody complex, is the ratio of(fluorescent signal from a complex comprising a polypeptide bound by aprimary antibody which in turn is bound to a binding agent labeled witha compound of the invention)/(fluorescent signal from a mixture of thepolypeptide, an isotype control primary antibody, and the labeledbinding agent).

“Degree of labeling” or “DOL” as used herein refers to the number of dyemolecules which are attached per target molecule (including but notlimited to polypeptide and polynucleotide). For example, a single dyemolecule per a polypeptide such as an antibody represents a 1.0 degreeof labeling (DOL). If more than one dye molecule, on average, reactswith and is crosslinked to a polypeptide such as an antibody, the degreeof labeling is greater than 1 and may further be a number other than awhole integer. The higher the number of DOL, the greater extent oflabeling.

“Intracellular” as used herein refers to the presence of a givenmolecule in a cell. An intracellular molecule can be present within thecytoplasm, attached to the cell membrane, on the surface of anorganelle, or within an organelle of a cell.

“Substrate” or “solid substrate” when used in the context of a reactionsurface refers to the material that certain interaction is assayed. Forexample, a substrate in this context can be a surface of an array or asurface of microwell. It may also be a solid such as a polymer whichdoes not form a specific shape but has attachment points on its surface.

The terms “wavelength of maximum excitation” and “maximal fluorescenceexcitation wavelength” are used herein interchangeably. These termsrefer to the maximum wavelength at which a fluorescent compound absorbslight energy which excites the dye to emit maximal fluorescence. Theterm “absorption maximal wavelength” as applied to a dye refers thewavelength of light energy at which the dye most effectively absorbsexcitation energy to fluoresce. A fluorescent dye has a “maximalfluorescence emission wavelength” which is the wavelength at which thedye most intensely fluoresces. When a single wavelength is referred tofor any dye, it refers to the maximal wavelength of excitation,absorption, or emission, according to the context of the term, forexample, an absorption wavelength refers to the wavelength at which thecompound has maximal absorption, and an emission wavelength refers tothe wavelength at which the dye most intensely fluoresces.

Compounds of the Invention:

The present invention provides compounds of Formula I:

Herein, F represents a fluorophore or fluorescent group. In general,fluorophores suitable for the compounds of the invention (“F” in formulaI) are derived from fluorescent compounds which have substitution sitesthat allow the attachment to the -(L)_(m)- or T groups. The corestructures of a number of fluorescent groups including those of theirsub-categories may be suitable as fluorophores. A fluorophore generallymay comprise a structure comprising a minimal number of atoms necessaryto form a fluorescent group belonging to a class of fluorescent groups.As an example, a coumarin fluorescent group comprises the core structureof formula A as set forth below:

As another example, a fluorescein fluorescent group comprises the corestructure of formula B as set forth below:

As still another example, rhodamine fluorescent groups have the corestructure of formula C as set forth below:

As yet another example, indocarbocyanine fluorescent groups may have thecore structure of formula D as set forth below:

Core structures for other classes of fluorescent groups can be readilydetermined by one of ordinary skill in the art using the aboveprinciple. One of skill can appreciate that the determination of afluorescent group core structure can be somewhat arbitrary because theclassification of fluorescent groups may be arbitrary by itself. A classof fluorescent groups, for example, may be sub-classified into differentsubclasses, wherein each subclass of fluorescent groups comprises one ormore substituents unique to the particular subclass of fluorescentgroups. Thus, there may be a core structure for each subclass offluorescent groups. For example, 7-aminocoumarin, shown below as formulaE, is the core structure for all 7-aminocoumarin derivatives, which arethemselves a subclass of fluorescent groups belonging to the moregeneral coumarin fluorescent groups that have the core structure offormula A.

The following table shows typical excitation and emission wavelengthsfor a number of common classes of fluorophores and core structures:

Fluorescent Typical Emission Group Typical Excitation WavelengthsWavelengths Coumarin 300-500 nm 350-550 nm Fluorescein 470-520 nm500-540 nm Rhodamine 480-640 nm 510-660 nm Cyanine 350-1200 nm  360-1250nm  Pyrene 350-490 nm 400-510 nm

In some embodiments, F may be a derived from cyanine fluorescent group,Cy fluorescent group, xanthene fluorescent group, Alexa Fluorfluorescent group, coumarin fluorescent group, pyrene fluorescent group,Bodipy fluorescent group, ATTO fluorescent group or DY fluorescentgroup. As commonly known in the field, cyanine fluorescent groups maycomprise various sub-categories of cyanine fluorescent groups including,but not limited to, indocarbocyanine fluorescent groups, oxacarbocyaninefluorescent groups, thiacarbocyanine fluorescent groups, azacarbocyaninefluorescent groups (azacyanine fluorescent groups), styrylcyanine groupand merocyanine fluorescent groups, merely by way of example. Similarly,xanthene fluorescent groups may include, but are not limited to,fluorescein and its derivatives and various rhodamine fluorescentgroups, for example.

Other nonlimiting examples of fluorescent compounds for use asfluorophores in the invention include Acridine orange, Acridine yellow,Alexa Fluor fluorescent groups, ATTO fluorescent groups, Bodipyfluorescent groups, Auramine O, Benzanthrone,9,10-Bis(phenylethynyl)anthracene, 5,12-Bis(phenylethynyl)naphthacene,Carboxyfluorescein diacetate, Calcein, Carboxyfluorescein,1-Chloro-9,10-bis(phenylethynyl)anthracene,2-Chloro-9,10-bis(phenylethynyl)anthracene, Coumarin, Cyanine, Cy2, Cy3,Cy3.5, Cy5, Cy5.5, Cy7, DyLight Fluor fluorescent groups, Fluorescein,2′,7′-dichlorodihydrofluorescein, Hilyte Fluor fluorescent groups, LDS751, Oregon Green, Perylene, Phycobilin, Phycoerythrin,Phycoerythrobilin, Pyrene, Rhodamine and Ruthenium(II)tris(bathophenanthroline disulfonate). These compounds and derivativesor radicals of these compounds may be used as fluorophores of theinvention.

Other examples of fluorescent groups which may be used in the presentinvention include but are not limited to4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine andderivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BODIPY™and its derivatives and analogs, Brilliant Yellow, cyanine fluorescentgroups such as Cy3 and Cy5 and other derivatives, coumarin andderivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151),7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin,5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride),fluorescein and derivatives such as 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine;4-methylumbelliferone, oxazine fluorescent groups such as Nile Blue andother analogs; pyrene and derivatives such as pyrene, pyrene butyrateand succinimidyl 1-pyrene butyrate; rosamine fluorescent groups,tetramethyl rosamine, and other analogs, rhodamine and derivatives suchas 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC) and thiazine fluorescentgroups such as methylene blue and analogs. Additional fluorophoresapplicable for use in the present are disclosed in US patent applicationNos. 2003/0165942, 2003/0045717, and 2004/0260093 and U.S. Pat. No.5,866,366 and WO 01/16375, both of which are incorporated herein byreference. Additional examples are described in U.S. Pat. No. 6,399,335,published U.S. patent application No. 2003/0124576, and The Handbook-‘AGuide to Fluorescent Probes and Labeling Technologies, Tenth Edition’(2005) (available from Invitrogen, Inc./Molecular Probes), all of whichare incorporated herein by reference.

Fluorescent groups of the invention may also include fluorescentproteins. Such fluorescent proteins known in the art include GFP and itsvarious derivatives, described e.g. in U.S. Pat. Nos. 5,625,048;5,777,079; 6,066,475; 6,319,669; 6,046,925; 6,124,128 and 6,077,707.Additional fluorescent proteins are Y66F, Y66H, EBFP, GFPuv, ECFP,AmCyan1, Y66W, S65A, S65C, S65L, S65T, EGFP, ZsGreen1, EYFP, ZsYellow1,DsRed, DsRed2, AsRed2, mRFP1 and HcRed1.

Many such fluorescent groups are commercially available and may be usedin the synthesis of compounds of the present invention. Commercialsources of reactive fluorescent groups include Invitrogen (MolecularProbes), AnaSpec, Amersham (AP Biotech), Atto-Tec, Dyomics, Clontech andSigma-Aldrich.

The fluorophore may be attached through a linking moiety -(L)_(m)- to ajoining moiety T. In general, linking moieties (generally represented by“L”) may be any group connecting two moieties, such as fluorophores,water soluble polymers and reactive groups to each other or to any othergroup included in the compound of the invention. Synthetic accessibilityand convenience may generally dictate the nature of each linking moiety.In some embodiments, a linking moiety is a group containing about 1-100atoms and formed of one or more chemical bonds selected such that thegroup is a stable moiety. In other embodiments, a linking moiety isformed of one or more carbon-hydrogen, carbon-nitrogen, carbon-oxygen,carbon-sulfur, carbon-phosphorus, nitrogen-hydrogen, sulfur-hydrogen,phosphorus-hydrogen, sulfur-oxygen, sulfur-nitrogen, sulfur-phosphorus,phosphorus-oxygen, phosphorus-nitrogen and oxygen-nitrogen bonds,wherein such bonds may be single, double, triple, aromatic andheteroaromatic bonds selected such that the linking moiety is stable. Alinking moiety can be, for example, a divalent alkyl radical.Alternatively, a linking moiety may be an alkyl group comprisingadditional ether, amine, amide, ester, sulfonyl, thioether, carboxamide,sulfonamide, hydrazide or morpholino, aryl and heteroaryl groups.

Linking moieties are generally formed of about 1-100 atoms. In someembodiments, linking moieties are formed of 1-50 non-hydrogen atoms aswell as additional hydrogen atoms. Such atoms may be, for example, C, N,O, P or S. In other embodiments, a linker moiety connecting two groupscomprises 1 to 50 consecutive bonds between the groups. Some linkermoieties may have 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 5 to 25,or 5 to 20 such consecutive bonds.

Non-limiting exemplary linking moieties are illustrated below:

In the above image, n represents a number of repeating methylene unitswhich can be varied such as to provide a desired length of the linker.Typically, n ranges from 1 to about 50. Some linkers will have an n of 1to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 5 to 30, 5 to 20, or 5 to 15.

Similarly, joining moieties (generally represented by “T”) may be anygroup connecting three or more distinct moieties such as fluorophores,water soluble polymers and reactive groups to each other or to any othergroup, such as a linker moiety, included in the compound of theinvention. In some embodiments, a joining moiety is a group containingabout 1-100 atoms and formed of one or more chemical bonds. The bondsmay be selected such that the group is a stable moiety. In otherembodiments, a joining moiety is formed of one or more carbon-hydrogen,carbon-nitrogen, carbon-oxygen, carbon-sulfur, carbon-phosphorus,nitrogen-hydrogen, sulfur-hydrogen, oxygen-hydrogen,phosphorus-hydrogen, sulfur-oxygen, sulfur-nitrogen, sulfur-phosphorus,phosphorus-oxygen, phosphorus-nitrogen and oxygen-nitrogen bonds,wherein such bonds may be single, double, triple, aromatic andheteroaromatic bonds selected such that the group is a stable moiety. Insome embodiments, T contains between 1 and 50 atoms, or alternativelybetween 1 and 40, 1 and 30, 1 and 20, or 1 and 10 atoms. T may be asingle atom such as N or C. T may also be a small cyclic group such as acarbocycle, a heterocycle, or an aromatic group. Nonlimiting examplesinclude substituted phenyl, naphthyl, tetrahydronaphthyl, indanyl,biphenyl, phenanthryl, anthryl, acenaphthyl, benzoimidazolyl,benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl,benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl,furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl,isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl,naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline,oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl,pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl,quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl,thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl,1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl,morpholinyl, thiomorpholinyl, dihydrobenzoimidazolyl,dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl,dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl. Joiningmoieties may be, for example, trivalent, tetravalent, pentavalent orhexavalent. Examples of joining moieties are depicted below:

The subscripts m and n of Formula I indicate the number of linkermoieties present and are independently integers ranging from 0 to 20.When m is 0, the linker moiety is understood to be absent and any twomoieties shown as attached to such a linker moiety are understood to beconnected through a bond. When n is 0, any substituent qualified by “n”will be understood to be absent.

Groups denoted as R₁, R₂, and R₃ of Formula I are groups of the formula(R)_(p)-(L)_(q)-. When multiple (R)_(p) (L)_(q)- groups are present in acompound, each R, L, p or q is independent of any other R, L, p or qgroup present in the same compound. Each p is generally an integerranging from 1 to 20. In some embodiments, p is 1. In other embodiments,p may range from 1 to 2, 3, 4, 5, 10 or 15. Each q is generally aninteger ranging from 0 to 20. In embodiments where p is 0, L isunderstood to be absent and any R group shown as attached to L isunderstood to be connected directly through a bond. Alternatively, p maybe 1. In other cases, q may range from 1 to 2, 3, 4, 5, 10 or 15.

“R” may be any group that confers a desirable functional property to thecompound of the invention. More specific embodiments of R groups will bediscussed below.

Compounds of the invention comprise at least one R which is a reactivegroup. A reactive group is a chemical moiety capable of reacting with areaction partner on a substrate or substrate molecule to form a covalentbond. A compound of the invention can be used to label a wide variety ofmolecules or substrates that contain a suitable reaction partner or arederivatized to contain a suitable reaction partner. “Reactive group” and“reaction partner” may refer to groups on a compound of the presentinvention, or to groups on a molecule to be labeled. Here, by way ofconvenience, but not limitation, a bond-forming group on a compound willgenerally be referred to as a reactive group and a bond-forming group onthe substrate molecule will generally be referred to as a reactionpartner. “Reaction substrate”, “substrate” and “reaction partner” areused interchangeably throughout this document.

The reactive group and its reaction partner may be an electrophile and anucleophile, respectively, that can form a covalent bond with or withouta coupling agent or catalyst. According to one embodiment, the reactivegroup is a photoactivatable group capable of reacting with a hydrocarbonmolecule upon ultraviolet photoactivation or photolysis. According toanother embodiment, the reactive group is a dienophile capable ofreacting with a conjugated diene via a Diels-Alder reaction. Accordingto yet another embodiment, the reactive group is a 1,3-diene capable ofreacting with a dienophile. According to still another embodiment, thereactive group is an alkyne capable of reacting with an azido functionalgroup to form a 1,2,3-triazole linkage. According to still anotherembodiment, the reactive group is a 2-(diphenylphosphino)benzoic acidmethyl ester capable of reacting with an azido functional group to forman amide linkage via so-called Staudinger reaction. Merely by way ofexample, examples of useful reactive groups, functional groups, andcorresponding linkages according to the present invention are listedbelow in Table 1.

TABLE 1 Examples of Reactive Groups, Functional Groups, and CovalentLinkages Reaction Partner/ Resulting Covalent Reactive Group SubstrateLinkage activated esters* amines/anilines Carboxamides acrylamidesThiols Thioethers acyl azides** amines/anilines Carboxamides acylhalides amines/anilines Carboxamides acyl halides Alcohols/phenolsEsters acyl nitriles Alcohols/phenols Esters acyl nitrilesamines/anilines Carboxamides aldehydes amines/anilines Imines aldehydesor Hydrazines Hydrazones ketones aldehydes or Hydroxylamines Oximesketones alkyl halides amines/anilines alkyl amines alkyl halides ThiolsThioethers alkyl halides alcohols/phenols Esters alkyl sulfonates ThiolsThioethers alkyl sulfonates carboxylic acids Esters alkyl sulfonatesalcohols/phenols Esters anhydrides alcohols/phenols Esters anhydridesamines/anilines Carboxamides aryl halides Thiols Thiophenols arylhalides Amines aryl amines aziridines Thiols Thioethers boronatesGlycols boronate esters epoxides Thiols Thioethers haloacetamides ThiolsThioethers halotriazines amines/anilines Aminotrizaines halotriazinesalcohols/phenols triazinyl ethers imido esters amines/anilines Amidinesisocyanates amines/anilines Ureas isocyanates alcohols/phenols Urethanesisothiocyanates amines/anilines Thioureas maleimides Thiols Thioethersphosphoramidites Alcohols phosphite esters silyl halides Alcohols silylethers sulfonate esters amines/anilines alkyl amines sulfonate estersThiols Thioethers sulfonate esters Alcohols Ethers sulfonyl halidesamines/anilines Sulfonamides sulfonyl halides phenols/alcohols sulfonateesters azide alkyne 1,2,3-triazole Cis-platinum guanosinePlatinum-guanosine complex *Activated esters, as understood in the art,generally have the formula —COΩ, where Ω is a good leaving group, suchas succinimidyloxy (—OC₄H₄O₂), sulfosuccinimidyloxy (—OC₄H₃O₂—SO₃H), or-1-oxybenzotriazolyl (—OC₆H₄N₃), for example; or an aryloxy group oraryloxy substituted one or more times by electron-withdrawingsubstituent(s), such as nitro, fluoro, chloro, cyano, trifluoromethyl,or combinations thereof, for example, used to form activated arylesters; or a carboxylic acid activated by a carbodiimide to form ananhydride or mixed anhydride —OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a)and R^(b), which may be the same or different, are independently C₁-C₆alkyl, C₁-C₆ perfluoroalkyl, or C₁-C₆ alkoxy; or cyclohexyl,3-dimethylaminopropyl, or N-morpholinoethyl. **Acyl azides can alsorearrange to isocyanates.

The reactive group may be one that will react with an amine, a thiol, ahydroxyl or an aldehyde. The reactive group may be an amine-reactivegroup, such as a succinimidyl ester (SE), for example, or athiol-reactive group, such as a maleimide, a haloacetamide, or amethanethiosulfonate (MTS), for example, or an aldehyde-reactive group,such as an amine, an aminooxy, or a hydrazide, for example.

The compounds of the invention also comprise at least one R which is awater-soluble polymer group. According to the invention, water solublepolymer groups may significantly reduce the intramolecular mobility ofthe fluorescent group core structure and may thus improve thefluorescent group's fluorescence quantum yield. Such groups may alsoconfer other properties to the compounds to which they are attached,such as improvements of the photostability of the fluorescent group,reduced fluorescent group aggregation for biomolecule labeling,increased staining specificity of fluorescently labeled biomolecules(such as antibodies); and reduced immunogenicity and antigenicity oflabeled biomolecules (such as antibodies) in vivo.

Each water soluble polymer group is generally a substantially unreactiveand water-soluble moiety sufficiently large to improve the fluorescenceproperties of a compound. The term “polymer” used in this context doesnot require the presence of strictly repeating units. A molecule ofsufficient molecular size and solubility but without repeating units isconsidered a “water soluble polymer group” for the purposes of theinvention.

Water soluble polymer groups include, but are not limited to, organicpolymers and biomolecules such as polypeptides and carbohydrates. Watersoluble polymers may comprise ether groups, hydroxyl groups, tertiaryamine groups, quaternized amine groups, and/or guanidine groups. Eachwater soluble polymer may be linear, branched, cyclic or a combinationthereof. Water soluble polymers of the invention may comprise a singlechain or alternatively one, two, three, four or more chains. Watersoluble polymers with one, two, three, four or more branches may beused. The compounds of the invention may comprise any number of watersoluble polymer groups. Generally, compounds of the invention compriseat least 1 water soluble polymer groups up to about 8 water solublepolymer groups. In one embodiment, a compound comprises at least 2 watersoluble polymer groups to about 8 water soluble polymer groups. Inanother embodiment, a compound comprises at least 3 water solublepolymer groups up to about 8 water soluble groups. Suitable molecularweights of each water soluble polymer group or, alternatively, of allwater soluble polymer groups in one compound may be about 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000,4500, 5000, 10000, 15000, 20000 Da or greater. In one embodiment, thewater soluble polymer group of the invention has a molecular weightbetween 450 and 5000 Da. In another embodiment, the water solublepolymer group of the invention has a molecular weight between about 800and about 3000 Da. In yet another embodiment, the combined molecularweight of all water soluble polymer groups within a compound is fromabout 450 to about 5,000 Da. In still another embodiment, the combinedmolecular weight of all water soluble polymer groups within a compoundis from about 1,000 to about 3,000 Da.

In one embodiment, the water soluble polymer of the invention is apolyalkylene oxide. Suitable polyalkylene oxides include polyethyleneglycol (PEG), polypropylene glycol (PPG), polyethyleneglycol-polypropylene glycol (PEG-PPG) copolymers, and N-substitutedmethacrylamide-containing polymers and copolymers. Various polyalkyleneoxides suitable for the present invention, as well as methods of makingand using them are described in the following references: U.S. Pat. Nos.5,637,749; 5,650,388; 5,298,643; 5,605,976; 5,567,422; 5,681,567;5,321,095; 5,349,001; 5,405,877; 5,234,903; 5,478,805; 5,324,844;5,612,460; 5,756,593; 5,686,110; 5,880,131; 6,824,782; 5,808,096;6,013,283; 6,251,382. Commercial sources of polyalkylene oxide reagentsinclude Sigma-Aldrich, Nanocs, Creative Biochem, Pierce, Enzon, Nektarand Nippon Oils and Fats. Exemplary polyalkylene oxide groups are shownbelow:

Polyalkylene oxides may be additionally substituted as necessary toconfer other desired properties to the polymer. Such modifications maycomprise, for example, chemical linkages that increase or decrease thechemical stability of the polymer, which would allow tuning of thechemical or biological stability of the half-life of the polymer. Insome cases, polyalkylene oxide molecules are terminated or “capped” withvarious groups. Examples of such groups are hydroxy, alkyl ether (e.g.methyl, ethyl, propyl ethers), carboxymethyl ether, carboxyethyl ether,benzyl ether, dibenzylmethylene ether or dimethylamine. A polyalkyleneoxide may have one of many possible terminals, including but not limitedto hydroxyl, methyl ether, ethyl ether, carboxymethyl ether, andcarboxyethyl ether. In one embodiment, a polyalkylene oxide is apolyethylene glycol polymer terminated with a methyl ether. Such a groupmay be referred to as an mPEG. An mPEG generally has the formula of—(CH₂CH₂O)CH₃, wherein n is the number of ethylene glycol units and isdetermined by the size of said mPEG.

Other suitable polymers include derivatives and conjugates ofpoly(2-hydroxyethyl methacrylate), polyhydroxypropyl methacrylamide,poly(styrene sulfonic acid), poly(vinyl alcohol), or poly(2-vinylN-methylpyridinium iodide).

In another embodiment, the water soluble polymer of the invention is acarbohydrate. Such carbohydrates include monosaccharides orpolysaccharides and may be, for example, soluble starch, glycogen,dextran, pectin, mannan, galactan, hydroxymethylcellulose,hydroxyethylcellulose and other derivatized celluloses. When the watersoluble polymer is a carbohydrate, at least 30% of the hydroxyl groupspresent in the carbohydrate may be masked as methyl ethers,sulfonatoalkyl ethers, and/or acetate esters.

In yet another embodiment, the water soluble polymer of the invention isa polypeptide. Suitable polypeptides may comprise, for example, serine,arginine, polylysine with modified epsilon amino groups, or cysteinicacid. Other examples of such polypeptides are disclosed, for example, inWO 2006/081249. It is contemplated that such polypeptides may be used asthe water soluble polymer of the invention.

Water soluble polymers of the invention also comprise combinations ofthe different classes described above. For example, such a water solublepolymer would be a polypeptide linked to a polyalkylene oxide moiety.

Water soluble polymers do not generally comprise any group or groupsthat are incompatible with the chemistry of the reactive group or groupsincluded in the compound of the invention. For example, a water solublepolymer should not comprise strong nucleophiles if a reactive group isan electrophile. In a more specific example, a water soluble polymershould not comprise primary or secondary amines if a reactive group isan N-hydroxysuccinimidyl ester. As another specific example, a watersoluble polymer should not comprise a thiol when a reactive group is amaleimide. Likewise, a water soluble polymer should generally notcomprise a strong electrophile if a reactive group is a nucleophile.However, a water soluble may comprise a minimal number of weaknucleophiles or a minimal number of weak electrophiles such that thechemistry of the reactive group is not significantly affected, or thestability of the compound of the invention is not affected duringstorage and handling. Examples of weak nucleophiles are hydroxyl groups,which are commonly present in carbohydrate molecules. Thus, in someembodiments, when a water soluble polymer is a carbohydrate molecule, atleast 30% of the hydroxyl groups are preferably masked as ethers, suchas methyl ether, and/or as esters, such as acetate esters. In otherembodiments, all of the hydroxyl groups may be masked as ethers and/oresters.

In general, additional substituents (“R”) of the compounds of theinvention may in some cases be groups such as sulfonate (—SO₃ ⁻),phosphonate (—PO₃ ²⁻), and ammonium groups. Herein, the term ammoniummeans NH₄ ⁺, a trialkylammonium, or a tetraalkylammonium. One of skillscan appreciate that an ionic group requires a counter ion to balance itscharge. For example, each negatively charged —SO₃ ⁻ or —PO₃ ²⁻ maynecessitate one or two cations to balance the negative charge. Likewise,a positively charged ammonium may require an anion to maintainneutrality. In general, the nature of the counter ion is not critical aslong as the counter ion does not lower the solubility of saidfluorescent group. In some embodiments, when a substituent is —SO₃ ⁻ or—PO₃ ²⁻, the counter ion is H⁺, Na⁺, K⁺ or an ammonium. In otherembodiments, when the substituent is ammonium, the counter ion ispreferably chloride, fluoride, bromide, sulfate, phosphate, acetate orthe like. Some fluorescent groups may intrinsically possess a positivecharge or negative charge. In such a case, the intrinsic charge may actas a counter ion. Alternatively, the intrinsic charge may require acounter ion for maintaining neutrality. The rule for selecting a counterion for any intrinsic charge is as previously described. In someembodiments of the invention, at least one sulfonate group is present(—SO₃ ⁻) and any necessary counter ion is selected from H⁺, Na⁺, K⁺ andan ammonium. For reason of simplicity, any dissociable counter ion orcounter ions for most of the fluorescent group structures depictedherein may not be shown.

Such substituents may increase a compound's water solubility and/or itsfluorescent quantum yield. However, a relatively high number of chargedgroups is generally not desirable because it would result in a highlycharged fluorophore, which on conjugation to a protein, for example, maysignificantly change the isoelectric point of the protein, thus possiblyaffecting the biological properties of the labeled protein. For example,an antibody labeled with a highly charged fluorescent molecule may showhigh background in staining. In some embodiments, the number of suchcharged water-soluble R groups is 0-4, or 0-3. Because the fluorescentgroup of the invention has at least one water soluble polymer group,which is also capable of increasing the water solubility and/or thequantum yield of the fluorescent group, the number of charged R groups,such as sulfonate groups, can be kept to a minimum, thereby minimizingthe loss of biological specificity of labeled proteins.

Each substituent R may be the same or different and may be selected fromhalogens, —OH, —NH₂, —SO₂NH₂, and any carbon-containing substituentscomprising 1 to about 15 carbon atoms and optionally at least one heteroatom. When present, the at least one hetero atom is preferably selectedfrom the group consisting of halogens, N, O, S, P and Si. In some cases,R may be a dialkylamine substituent such as, for example, diethylamineor dimethylamine. When R is a carbon-containing substituent, it mayassume any structure or conformation, including, for instance, alkyl,cycloalkyl, alkenyl or alkynyl.

Compounds of the invention may adopt a variety of configurations.Exemplary configurations of specific configurations are shown in Table 2below:

TABLE 2 Exemplary configurations of compounds of formula I R₁ R₂ R3 F(L)_(m) (R)_(p) WSP (L)_(q) (R)_(p) RG (L)_(q) (R)_(p) (L)_(q) n TCoumarin, alkylene, PEG, MW alkylene, Amine- alkylene, 0 trivalentunsubstituted m = 0 or 300-800 q = 0 or reactive q = 0 or atom or 1 1 1group such as N or CH Thiol- reactive Alcohol- reactive PEG, MW Amine-800-2000 reactive Thiol- reactive Alcohol- reactive PEG, MW Amine-2000-5000 reactive Thiol- reactive Alcohol- reactive Coumarin, alkylene,PEG, MW alkylene, Amine- alkylene, 0 trivalent 1 additional m = 0 or300-800 q = 0 or reactive q = 0 or atom or substitution 1 1 1 group(alkyl, halo, such as amino, hydroxyl N or CH or sulfonyl) Thiol-reactive Alcohol- reactive PEG, MW Amine- 800-2000 reactive Thiol-reactive Alcohol- reactive PEG, MW Amine- 2000-5000 reactive Thiol-reactive Alcohol- reactive Coumarin, alkylene, PEG, MW alkylene, Amine-alkylene, 0 trivalent 2 additional m = 0 or 300-800 q = 0 or reactive q= 0 or atom or substitutions 1 1 1 group (alkyl, halo, such as amino,hydroxyl N or CH or sulfonyl) Thiol- reactive Alcohol- reactive PEG, MWAmine- 800-2000 reactive Thiol- reactive Alcohol- reactive PEG, MWAmine- 2000-5000 reactive Thiol- reactive Alcohol- reactive Coumarin,alkylene, PEG, MW alkylene, Amine- alkylene, 0 trivalent 3 additional m= 0 or 300-800 q = 0 or reactive q = 0 or atom or substitutions 1 1 1group (alkyl, halo, such as amino, hydroxyl N or CH or sulfonyl) Thiol-reactive Alcohol- reactive PEG, MW Amine- 800-2000 reactive Thiol-reactive Alcohol- reactive PEG, MW Amine- 2000-5000 reactive Thiol-reactive Alcohol- reactive Coumarin, alkylene, PEG, MW alkylene, Amine-alkylene, 0 trivalent substituted with m = 0 or 300-800 q = 0 orreactive q = 0 or atom or fused ring 1 1 1 group (optionally such assubstituted with N or CH another Thiol- reactive Alcohol- reactive PEG,MW Amine- 800-2000 reactive Thiol- reactive Alcohol- reactive PEG, MWAmine- 2000-5000 reactive Thiol- reactive Alcohol- reactiveIndocarbocyanine alkylene, PEG, MW alkylene, Amine- alkylene, 0trivalent fluorescent m = 0 or 300-800 q = 0 or reactive q = 0 or atomor group, 1 1 1 group unsubstituted such as N or CH Thiol- reactiveAlcohol- reactive PEG, MW Amine- 800-2000 reactive Thiol- reactiveAlcohol- reactive PEG, MW Amine- 2000-5000 reactive Thiol- reactiveAlcohol- reactive Indocarbocyanine alkylene, PEG, MW alkylene, Amine-alkylene, 0 trivalent fluorescent m = 0 or 300-800 q = 0 or reactive q =0 or atom or group, 1 1 1 group With additional such as substitution Nor CH (alkyl, halo, amino, hydroxyl or sulfonyl) Thiol- reactiveAlcohol- reactive PEG, MW Amine- 800-2000 reactive Thiol- reactiveAlcohol- reactive PEG, MW Amine- 2000-5000 reactive Thiol- reactiveAlcohol- reactive Rhodamine alkylene, PEG, MW alkyl, Amine- alkylene, 0trivalent fluorescent group m = 0 or 300-800 q = 0 or reactive q = 0 oratom or 1 1 1 group such as N or CH Thiol- reactive Alcohol- reactivePEG, MW Amine- 800-2000 reactive Thiol- reactive Alcohol- reactive PEG,MW Amine- 2000-5000 reactive Thiol- reactive Alcohol- reactive Rhodaminealkylene, PEG, MW alkylene, Amine- alkylene, 0 trivalent fluorescent m =0 or 300-800 q = 0 or reactive q = 0 or atom or group, 1 1 1 groupadditional such as substitutions N or CH (alkyl, halo, amino, hydroxylor sulfonyl) Thiol- reactive Alcohol- reactive PEG, MW Amine- 800-2000reactive Thiol- reactive Alcohol- reactive PEG, MW Amine- 2000-5000reactive Thiol- reactive Alcohol- reactive Fluorescein alkylene, PEG, MWalkylene, Amine- alkylene, 0 trivalent fluorescent group m = 0 or300-800 q = 0 or reactive q = 0 or atom or 1 1 1 group such as N or CHThiol- reactive Alcohol- reactive PEG, MW Amine- 800-2000 reactiveThiol- reactive Alcohol- reactive PEG, MW Amine- 2000-5000 reactiveThiol- reactive Alcohol- reactive Fluorescein alkylene, PEG, MWalkylene, Amine- alkylene, 0 trivalent fluorescent m = 0 or 300-800 q =0 or reactive q = 0 or atom or group, 1 1 1 group additional such assubstitutions N or CH (alkyl, halo, amino, hydroxyl or sulfonyl) Thiol-reactive Alcohol- reactive PEG, MW Amine- 800-2000 reactive Thiol-reactive Alcohol- reactive PEG, MW Amine- 2000-5000 reactive Thiol-reactive Alcohol- reactive Pyrene alkylene, PEG, MW alkylene, Amine-alkylene, 0 trivalent m = 0 or 300-800 q = 0 or reactive q = 0 or atomor 1 1 1 group such as N or CH Thiol- reactive Alcohol- reactive PEG, MWAmine- 800-2000 reactive Thiol- reactive Alcohol- reactive PEG, MWAmine- 2000-5000 reactive Thiol- reactive Alcohol- reactive Pyrene,alkylene, PEG, MW alkylene, Amine- alkylene, 0 trivalent additional m =0 or 300-800 q = 0 or reactive q = 0 or atom or substitutions 1 1 1group (alkyl, halo, such as amino, hydroxyl N or CH or sulfonyl) Thiol-reactive Alcohol- reactive PEG, MW Amine- 800-2000 reactive Thiol-reactive Alcohol- reactive PEG, MW Amine- 2000-5000 reactive Thiol-reactive Alcohol- reactive In the above Table, “WSP” signifies a watersoluble polymer group, while “RG” represents a reactive group.

In one embodiment, the compound of formula I comprises a fluorophorewhich is a xanthene fluorescent group, a coumarin fluorescent group, apyrene fluorescent group or a cyanine fluorescent group. In someembodiments, the fluorophore is a coumarin fluorescent group. Such afluorophore may have the formula

where one moiety of R_(a), R_(b), R_(c), R_(d), R_(e) and R_(f) is abond connecting the fluorophore to a moiety -(L)_(m)- or a moiety

as indicated in Formula I. The remaining moieties R_(a), R_(b), R_(c),R_(d), R_(e) and R_(f) have the formula (R)_(p)-(L)_(q)-, where R, L, pand q are as previously defined. For example, any adjacent pair ofR_(a), R_(b), R_(c), R_(d), R_(e) and R_(f) may join to form a 5- or6-membered, saturated or unsaturated ring that may optionally compriseadditional heteroatoms in the ring as well as additional R substituents.In some embodiments, such R substituents are SO₃ ⁻, sulfonamido, halo,hydroxy, amino or alkyl groups.

In another embodiment, the fluorophore is a compound of the formula:

wherein

connects said fluorophore to said moiety -(L)_(m)- or said moiety

R₄, R₅, and R₆ are each independently (R)_(p)-(L)_(q)-; each R of R₄,R₅, R₆ is independently i) a reactive group capable of forming acovalent bond upon reacting with a reaction partner; ii) a water solublepolymer group; iii) an alkyl group, a trifluoroalkyl group, a halogengroup, a sulfonate group or a sulfonamido group; or iv) —H; each L ofR₄, R₅ and R₆ is independently a linking moiety formed of one or morechemical bonds and containing about 1-100 atoms; each p of R₄, R₅, andR₆ is independently an integer ranging from 1 to 20; each q of R₄, R₅,and R₆ is independently an integer ranging from 0 to 20; and a, b, and care independently 0, 1, 2, or 3.

In one embodiment, T is a trivalent moiety comprising one carbon atomsuch as

Alternatively, T may be a trivalent nitrogen atom. For example,

in Formula I may have the formula

When R₁ is a water soluble polymer, said water-soluble polymer may havea molecular weight of greater than about 300 Da. Alternatively, themolecular weight may be greater than 800 Da, or it may range from about800 Da to about 3000 Da. R₁, may, for example, comprise a water solublegroup such as a polyethylene glycol group. In some embodiments, themolecular weight of the polyethylene glycol group is between 450 and5000 Da.

When R₂ is a reactive group, the reactive group may form a covalentbond, for example, with amino, sulfhydryl or hydroxy nucleophiles. Insome embodiments, the reactive group is an isothiocyanate, anisocyanate, a monochlorotriazine, a dichlorotriazine, ahalogen-substituted pyridine, a halogen-substituted diazine, aphosphoramidite, a maleimide, an aziridine, a sulfonyl halide, an acidhalide, a hydroxysuccinimide ester, a hydroxysulfosuccinimide ester, animido ester, a hydrazine, an azidonitrophenyl, an azide, an alkyne, a3-(2-pyridyl dithio)-propionamide, a glyoxal or an aldehyde. In someembodiments, the reactive group is an N-hydroxysuccinimide ester.

In another aspect of the invention, a compound is provided having amaximal fluorescence excitation wavelength wherein the compound has astructure of Formula II:F—Y=Ψ  Formula II

wherein:

F is a moiety having the structure:

Z⁻ is a counterion.

Y is a bridge unit permitting electron delocalization between F and □.Suitable moieties are well known in the art in the context of cyaninefluorescent groups. Generally, a Y group will allow electrondelocalization between said two structures. For example, Y may be amethine unit or may alternatively be a polymethine unit (tri-, penta- orheptamethine) optionally incorporating one or more 4, 5, or 6-memberedrings. Additional substitutions with R groups of the invention are alsocontemplated. For example, Y may comprise an R group which is a watersoluble polymer, a reactive group or an additional substituent asdefined above. For example, alkyl, SO₃ ⁻ and halogen are all possiblesubstituents present as part of the Y group.

In some embodiments, Ψ is a moiety having one of the followingstructures:

X₁ and X₄ are independently

X₁ and X₄ may or may not be additionally substituted.

X₂ and X₃ are independently

The elements a and b are independently 0, 1, 2, or 3.

The element b′ is 0, 1 or 2.

In some embodiments, when Ψ is Formula 1, and the maximal fluorescenceexcitation wavelength of the compound is less than 660 nm, then R₅ andR₆ are independently (R)_(p) (L)_(q)-, wherein R₅ and R₆ are notcombinable to form a substituted ring; In other embodiments of theinvention, when Ψ is Formula 1, and the maximal fluorescence excitationwavelength of the compound is equal to or greater than 660 nm, or Ψ isother than Formula 1, then R₅ and R₆ are independently (R)_(p)-(L)_(q)-,or R₅ and R₆ are combinable to form a cyclic moiety which isunsubstituted or substituted by one or more (R)_(p)-(L)_(q)-. The cyclicmoiety so formed is a 5, 6, or 7 membered ring with is carbocyclic orheterocyclic, and in some embodiments, substituted by one or morereactive groups and/or one or more water soluble polymers.

In yet other embodiments, when Ψ is Formula 1, and the maximalfluorescence excitation wavelength of the compound is less than 655 nm,then R₅ and R₆ are independently (R)_(p) (L)_(q)-, wherein R₅ and R₆ arenot combinable to form a substituted ring; In other embodiments of theinvention, when Ψ is Formula 1, and the maximal fluorescence excitationwavelength of the compound is equal to or greater than 655 nm, or Ψ isother than Formula 1, then R₅ and R₆ are independently (R)_(p)-(L)_(q),or R₅ and R₆ are combinable to form a cyclic moiety which isunsubstituted or substituted by one or more (R)_(p) (L)_(q)-.

In various embodiments, Y is:

wherein when C is present, it is a five- or six-membered cyclic group.

R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are each independently (R)_(p)-(L)_(q)-.

Each R of each (R)_(p)-(L)_(q)- of the compound is independently i) areactive group capable of forming a covalent bond upon reacting with areaction substrate; ii) a water soluble polymer group; iii) an alkylgroup, an aryl group, an alkylamino group, a dialkylamino group, analkoxy group, a trifluoroalkyl group, a halogen group, a sulfonyl group,a sulfonate group or a sulfonamido group; or iv) —H.

Each L of each (R)_(p)-(L)_(q)- of the compound is independently alinking moiety formed of one or more chemical bonds and containing about1-100 atoms.

Each p of each (R)_(p)-(L)_(q)- is independently an integer of about 1to about 20.

Each q of each (R)_(p)-(L)_(q)- of R₁ or R₂ is independently an integerof 0 to about 20.

Each q of each (R)_(p)-(L)_(q)- of R₃, R₄, R₅, R₆, or R₇, isindependently an integer of 1 to about 20.

The element c is 0 or 1.

The element d is 0 or 1.

At least one R of the (R)_(p) (L)_(q)- of the compound is a reactivemoiety; and at least one R of the (R)_(p)-(L)_(q)- of the compound is awater-soluble polymer.

In some embodiments, when at least two adjacent R₁, and/or two adjacentR₂ are present, the two adjacent R₁, and/or the two adjacent R₂ arecombinable to form a 6-membered ring which is unsubstituted orsubstituted by one or more (R)_(p)-(L)_(q)-. In some other embodiments,when the two adjacent R₁, and/or the two adjacent R₂ are combinable toform a 6-membered ring, the ring so formed is aromatic. In someembodiments, two adjacent (R₂)_(b) and the atoms in ring B to which itis attached are combined to form a carbocyclic ring, optionallyadditionally substituted with groups that may further increase thesolubility of the fluorescent group. In some embodiments, two adjacent(R₂)_(b) and the atoms in ring B to which it is attached are combined toform a carbocyclic ring which is aromatic and optionally additionallysubstituted with groups that may further increase the solubility of thefluorescent group. In some embodiments, two adjacent (R₁)_(b) and theatoms in ring A to which it is attached are combined to form acarbocyclic ring, and optionally additionally substituted with groupsthat may further increase the solubility of the fluorescent group. Insome embodiments, two adjacent (R₁)_(b) and the atoms in ring A to whichit is attached are combined to form a carbocyclic ring which isaromatic, which is optionally additionally substituted with groups thatmay further increase the solubility of the fluorescent group.

The element c may be 0 or 1. When c is 1, the nitrogen atom to which R₃is attached is positively charged. Similarly, d may be 0 or 1.

In various embodiments, at least one R of R₁ and R₂ is a charged moiety.In some embodiments, at least one R of R₁ and R₂ comprises a sulfonategroup or a phosphonate group. In yet other embodiments, each R of R₁ andR₂ comprises a sulfonate group or a phosphonate group.

In various embodiments of the invention, X₂ and X₃ are independently

In related embodiments, X₂ and X₃ are alkyl groups such as methyl orethyl. In some embodiments, X₂ and X₃ are

In some embodiments, X₂ and X₃ are:

wherein R₅ comprises a reactive group or a water-soluble polymer.

The compound of formula II contains at least one reactive group and awater-soluble polymer. These groups may be attached at any positionshown in formula II. For example, a reactive group or a water-solublepolymer may be attached to ring A or ring B. Alternatively, the reactivegroup or the water-soluble polymer may substitute the nitrogen atomsindicated in formula II, e.g. R₃ could be a reactive group and R₄ couldbe a water-soluble polymer, optionally attached through L linkermoieties. Furthermore, reactive groups and/or water-soluble polymers mayalso be attached to the X₂ and/or X₃ positions in formula II, or may bepart of the linker structure Y.

In some cases, it may be desirable to include at least one R substituentwhich is a charged moiety on ring A or one ring of B, D, E, F, and G orboth ring A and one ring of B, D, E, F and G such as to increase thesolubility of the compound of formula II. Such a moiety may be asulfonate group or any other group as previously described. Similarsubstitution can be made as part of the groups R₃, R₄, X₂ and X₃, forexample by attaching a linker moiety which is substituted with asulfonate, phosphonate or other charged group.

In various embodiments of the invention, the water-soluble polymer is apolyalkylene oxide. In other embodiments, the water-soluble polymer is apolyethylene oxide. In yet other embodiments, the water-soluble polymeris a carbohydrate. In some other embodiments, the water-soluble polymeris a polypeptide. In various embodiments, the water-soluble polymer hasa molecular weight ranging from about 800 to about 3000. In someembodiments, the water-soluble polymer has a molecular weight of greaterthan 300. In other embodiments, the water-soluble polymer has amolecular weight of greater than 800.

In some embodiments the compound of Formula II comprises at least onereactive group and at least two water-soluble polymers.

In some embodiments of the compound of Formula II, the compound has theformula:

In various embodiments, the compound of Formula II has the formula:

wherein c is 1; d is 1; at least one R of R₃ and R₄ is a reactive groupcapable of forming a covalent bond upon reacting with a reactionsubstrate; and at least one R of R₃ and R₄ is a water soluble polymergroup.

In other embodiments, the compound has the formula:

wherein c is 1; and d is 1; at least one R of R₃, R₄ and R₅ is areactive group capable of forming a covalent bond upon reacting with areaction substrate; and at least one R of R₃, R₄ and R₅ is a radical ofa water-soluble polymer. In another embodiment, c is 1; d is 1; one R ofR₃, R₄ and R₅ is a reactive group; and at least two R of R₃, R₄ and R₅are a radical of a water-soluble polymer. In one embodiment, Y isselected such that the absorption maximal wavelength is about 550 nm,about 650 nm, or about 750 nm.

In yet other embodiments, the compound has the formula:

wherein c is 1; d is 1;

one R of R₃ and R₄ is a reactive group capable of forming a covalentbond upon reacting with a reaction substrate; and one R of R₃ and R₄ isa water soluble polymer group.

In some other embodiments, the compound has the formula:

wherein c is 1; d is 1;

at least one R of R₃, R₄ and R₅ is a reactive group capable of forming acovalent bond upon reacting with a reaction substrate; and at least oneR of R₃, R₄ and R₅ is a radical of a water-soluble polymer.

In other embodiments, the compound has the formula:

wherein c is 1; d is 1;

at least one R of R₃, R₄ and R₅ is a reactive group capable of forming acovalent bond upon reacting with a reaction substrate; and at least oneR of R₃, R₄ and R₅ is a radical of a water-soluble polymer. In anotherembodiment,

In another embodiment, the compound has the structure:

wherein, c is 1; d is 1; one R of R₃, R₄ and R₅ is a reactive group; andat least two R of R₃, R₄ and R₅ each comprise a radical of awater-soluble polymer. In one embodiment, Y is selected such that theabsorption maximal wavelength is about 580 nm, about 680 nm, or about790 nm.

In yet another embodiment, the compound has the structure:

wherein, c is 1; d is 1; one R of R₃, R₄ and R₅ is a reactive group; andat least two R of R₃, R₄ and R₅ are each a radical of water-solublepolymers. In one embodiment, Y is selected such that the absorptionmaximal wavelength is around 560 nm, around 660 nm, or around 770 nm.

For compounds of Formula II, it is well known to a person of skill inthe art to select the bridging unit Y, in combination with a specificembodiment of a moiety of Formula F, in combination with a specificembodiment of moiety Y, and in combination with specific substituentssuch that the maximal fluorescence excitation wavelength of the compoundranges from about 350 to about 1200 nm. Standard analysis well known inthe art is used to obtain the absorption and emission spectra of thedyes in order to ascertain the absorption maximal wavelength and themaximal fluorescence emission wavelength. Any combinations of the abovedescribed groups may be used to form a compound of the invention with amaximal fluorescence excitation wavelength of about 550 nm, about 560nm, about 570 nm, about 580 nm, about 590 nm, about 600 nm, about 610nm, about 620 nm, about 615 nm, about 620 nm, about 625 nm, about 630nm, about 635 nm, about 640 nm, about 650 nm, about 655 nm, about 660nm, about 665 nm, about 670 nm, about 675 nm, about 680 nm, about 685nm, about 690 nm, about 695 nm, about 700 nm, about 705 nm, about 710nm, about 715 nm, about 720 nm, about 725 nm, about 730 nm, about 735nm, about 740 nm, about 745 nm, about 750 nm, about 755 nm, about 760nm, about 765 nm, about 770 nm, about 775 nm, about 780 nm, about 790nm, about 800 nm, about 810 nm, about 820 nm, about 830 nm, about 840nm, about 850 nm, about 860 nm, about 870 nm, about 880 nm, about 890nm, about 900 nm, about 910 nm, about 920 nm, about 930 nm, about 940nm, about 950 nm, about 960 nm, about 970 nm, about 980 nm, about 990nm, about 1000 nm, about 1020 nm, about 1040 nm, about 1060 nm, about1080 nm, about 1100 nm, about 1120 nm, about 1140 nm, about 1160 nm,about 1180 nm, or about 1200 nm. The moieties described above may beused in any combination. The maximal fluorescence excitation wavelengthof the dye is typically the wavelength at which the dye has a maximalabsorption or maximal optical density, which excites the dye tofluoresce. In some embodiments, the compound of Formula II has anabsorption maximal wavelength at >670 nm. In one embodiment, theabsorption maximal wavelength is at >700 nm. According to anotherembodiment, the absorption maximal wavelength is at >800 nm.

A person of skill can also combine select the bridging unit Y, incombination with a specific embodiment of a moiety of Formula F, incombination with a specific embodiment of moiety Y, and in combinationwith specific substituents such that one obtains a compound of FormulaII having a maximal fluorescence emission wavelength of about 550 nm,about 560 nm, about 570 nm, about 580 nm, about 590 nm, about 600 nm,about 610 nm, about 620 nm, about 615 nm, about 620 nm, about 625 nm,about 630 nm, about 635 nm, about 640 nm, about 650 nm, about 655 nm,about 660 nm, about 665 nm, about 670 nm, about 675 nm, about 680 nm,about 685 nm, about 690 nm, about 695 nm, about 700 nm, about 705 nm,about 710 nm, about 715 nm, about 720 nm, about 725 nm, about 730 nm,about 735 nm, about 740 nm, about 745 nm, about 750 nm, about 755 nm,about 760 nm, about 765 nm, about 770 nm, about 775 nm, about 780 nm,about 790 nm, about 800 nm, about 810 nm, about 820 nm, about 830 nm,about 840 nm, about 850 nm, about 860 nm, about 870 nm, about 880 nm,about 890 nm, about 900 nm, about 910 nm, about 920 nm, about 930 nm,about 940 nm, about 950 nm, about 960 nm, about 970 nm, about 980 nm,about 990 nm, about 1000 nm, about 1020 nm, about 1040 nm, about 1060nm, about 1080 nm, about 1100 nm, about 1120 nm, about 1140 nm, about1160 nm, about 1180 nm, about 1200 nm, about 1220 nm, about 1240 nm, orabout 1250 nm. The moieties described above may be used in anycombination.

According to one embodiment, the wavelength of the absorption maximum ofa compound of formula II is at least greater than 655 nm.

According to one embodiment, the wavelength of the absorption maximum ofa compound of formula II is at least greater than 670 nm. In stillanother embodiment, the wavelength of the absorption maximum of acompound of formula II is at least greater than 670 nm. In yet anotherembodiment, the wavelength of the absorption maximum of a compound offormula II is at least greater than 700 nm.

In another aspect of the invention, a substituted cyanine dye isprovided which comprises one or more water soluble polymer groups,wherein the cyanine dye has a maximal fluorescence excitation wavelengthof equal to or greater than about 660 nm. In some embodiments, thesubstituted cyanine dye comprises at least one reactive group. In someembodiments, the substituted cyanine dye is substituted by a non-spiromoiety.

In a further aspect of the invention, a substituted cyanine dye isprovided which comprises one or more water soluble polymer groups,wherein the cyanine dye has an absorption maximal wavelength of equal toor greater than about 660 nm. In some embodiments, the substitutedcyanine dye comprises at least one reactive group. In some embodiments,the substituted cyanine dye is substituted by a non-spiro moiety.

Exemplary Structures of Compounds of the Formula II are Shown Below inTable 3

TABLE 3 Exemplary structures of compounds of the formula IIλ_(abs)/λ_(cm) (nm) Dye No. Structure (H₂O) 1

457/ 2

550/570 3

550/570 4

550/570 5

550/570 6

550/570 7

550/570 8

550/570 9

650/665 10

650/665 11

650/665 12

650/665 13

650/665 14

660/680 15

650/665 16

663/690 17

752/778 18

750/775 19

675/694 20

750/775 21

22

768/788 23

24

635/642 25

26

497/513 27

555/565 28

650/665 29

750/770 30

660/675 31

770/790 32

680/700 33

790/810 *For simplicity, counter ions are not shown.

Dye compounds of formula II have excellent solubility and superiorfluorescence brightness and photostability. In particular, compounds offormula II having an absorption maximal wavelength greater than about655 nm, typically referred to as near-IR dyes, showed major advantagesover prior art dyes of similar wavelengths. The water-soluble polymersubstituents drastically improved the performance of the near-IR dyes ofthe invention, resulting in unprecedented fluorescence brightness (SeeFigures). The water-soluble polymer substituents dramatically improvedthe stability of the near-IR dyes (See Figures). The stability ofnear-IR dyes has been a problem, which demands extreme care in storageand handling and therefore limits the use of the dyes.

The invention also provides a compound of the formula III:

Z may be —H, alkyl or a substituent such as —CF₃, or —CN. Alternatively,Z is:

Compounds of formula III generally belong to the class of xanthenefluorescent groups. The choice of X₁, X₂ and Z further determineswhether the compound belongs to the class of fluorescein fluorescentgroups (X₁ is ═O, X₂ is —OH, Z is substituted phenyl), or rhodaminefluorescent groups (X₁ is ═NH₂ ⁺ or ═NR₆R₇ ⁺, X₂ is —NH₂ or —N₈R₉, Z issubstituted phenyl), or rhodol groups (X₁ is ═O, X₂ is —NH₂ or —NR₈R₉).R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are each independently (R)_(p)(L)_(q)-, where L, p and q are as generally defined elsewhere in thisdocument. R is as previously defined, with the addition that R alsoincludes moieties which have both a water-soluble polymer and a reactivegroup, for example joined by a joining moiety. The variables c, d and eindicate the numbers of R₃, R₄ and R₅ substituents on the phenyl ring ingroup Z and may be 0, 1, 2 or 3, such that the sum of c, d and e is lessor equal to 5.

Each compound of formula III comprises a reactive moiety as well as awater-soluble polymer. Therefore, at least one R of R₁, R₂, R₃, R₄, R₅,R₆ and R₇ is a reactive moiety while at least one R of R₁, R₂, R₃, R₄,R₅, R₆ and R₇ is a water-soluble polymer group.

In some embodiments, the compound of formula III belongs to the class offluorescein fluorescent groups and X₁ and X₂ are ═O and —OH,respectively, while Z is a phenyl group substituted with at least onemoiety of the formula (R)_(p) (L)_(q)-. In a related embodiment, Zcomprises at least one R which is a water soluble polymer and at leastone R which is a reactive group.

In other embodiments, the compound of formula III belongs to the classof rhodamine fluorescent groups, wherein X₁ is ═NH₂ ⁺ or ═NR₆R₇ ⁺, X₂ is—NH₂ or —NR₈R₉, and Z is a phenyl moiety substituted with at least one(R)_(p)-(L)_(q)- moiety (at least one of c, d or e is not 0). By way ofexample, X₂ may be an amino group which is optionally substituted withone or two groups such as alkyl. In such cases, X₂ may be, for instance,a dimethylamino, diethylamino, methylamino or ethylamino substituent. Ifneither R₁ or X₂ comprises a water-soluble polymer group or reactivegroup, group Z may be substituted with at least one reactive group andat least one water soluble polymer.

In other cases, R₁ and X₂ taken together form a carbocyclic orheterocyclic ring fused to ring A in formula III. In relatedembodiments, R₂ and X₁ taken together form a carbocyclic or heterocyclicring fused to ring B in formula III. Such fused rings may additionallybe substituted with additional R groups, for example SO₃ ⁻, alkyl, oreven water soluble polymer groups or reactive groups. The rings soformed by combining one of R₆, R₇, R₈ and R₉ with a neighboring R₁,and/or R2 are unsaturated or saturated and may be unsubstituted orsubstituted by one or more (R)_(p)-(L)_(q)-.

When group Z is —H, —CN, or —CF₃, one R of R₁, X₁, R₂ or X₂ comprises awater soluble polymer group while another R of R₁, X₁, R₂ or X₂comprises a reactive group.

In some embodiments, water soluble groups comprise a polyalkylene oxideattached to the core structure of formula III via a linker moiety orjoining moiety. It is possible for either R₃, R₄ or R₅ to be a joiningmoiety to which both a water soluble polymer and a reactive group areconnected via independent linker moieties.

Exemplary compounds of formula III are illustrated in Table 4.

TABLE 4 Exemplary Xanthene Compounds* λ_(abs)/λ_(cm) (nm) Dye No.Structure (H₂O) 34

494/520 35

36

501/524 37

38

39

520/546 40

41

42

540/565 43

44

45

46

47

48

488/515 49

494/520 *For simplicity, counter ions are not shown.

In one embodiment of the invention, the compound is a coumarinfluorescent group having the formula IV shown below:

wherein R₁, R₂, R₃, R₄, R₅ and R₆ are (R)_(p)-(L)_(q)- as definedpreviously, provided that at least one R of R₁, R₂, R₃, R₄, R₅ and R₆comprises a water soluble polymer and at least one other R of R₁, R₂,R₃, R₄, R₅ and R₆ comprises a reactive group.

According to one embodiment, R₅ is —OH or —NR₇R₈ where R₇ and R₈ areindependently H, an alkyl group optionally comprising at least oneheteroatom, a reactive group, a water-soluble polymer group, or asulfonate group. Alternatively, R₇ in combination with R₈ forms asubstituted or unsubstituted 5- or 6-membered ring that optionallycomprises at least one heteroatom. In another aspect of the invention,R₇ in combination with R₄, and/or R₈ in combination with R₆ form asaturated or unsaturated 5- or 6-membered ring that may be substitutedor unsubstituted, and/or may fuse with another 5-membered heterocyclicring, optionally substituted with additional R groups.

According to another embodiment, R₃ and R₆ are each H; R₂ and R₁,independently comprise R groups as previously defined; R₁, mayadditionally comprise an R group which is aryl, where the aryloptionally comprises at least one heteroatom selected from halogens, N,P, O, S and Si, and optionally comprises one or more additional Rsubstituents.

Selected coumarin dyes according to the invention are listed in Table 5.

TABLE 5 List of selected coumarin-based fluorescent groups according tothe invention* λ_(abs)/λ_(cm) (nm) Dye No. Structure (H₂O) 50

353/442 51

346/442 52

416/465 53

430/545 54

350/440 *For simplicity, any counter ions for the structures are notshown.

The invention also provides a compound of the formula V:

In Formula V, R₁, R₂, R₃, and R₄ are each independently(R)_(p)-(L)_(q)-, provided that at least one R of R₁, R₂, R₃ and R₄ is areactive moiety; and at least one R of R₁, R₂, R₃ and R₄ is awater-soluble polymer group. The variables a, b, c and d areindependently 0, 1, 2 or 3. Generally, the sum of a, b, c and d is 2, 3or 4. In one embodiment, the sum of a, b, c and d is 4. For example, R₁,R₂ and R₃ may each comprise water soluble polymer groups, such that each(R)_(p)-(L)_(q)- group may independently have the formula—SO₂NH(CH₂CH₂O)_(n)CH₃, where n is from about 3 to about 30, or fromabout 7 to about 24. In this embodiment, R₄ comprises an R which is areactive group such as an activated ester of a carboxylic acid.

In other embodiments, L may comprise a C1-C8 N-alkyl sulfonamide or aC2-C10 N,N-dialkyl sulfonamide group linking either a reactive group ora water soluble polymer group to the pyrene moiety in formula V, suchthat each sulfonamide is covalently linked to the pyrene carbon via S—Cbond and where each alkyl portion optionally comprises at least one O.

In another embodiment, R₁, R₂ and R₃ comprise substituents such assulfonate groups and R₄ is a joining moiety to which both a watersoluble polymer and a reactive group are connected via independentlinker moieties.

Exemplary compounds of the Formula V are listed in Table 6.

TABLE 6 Exemplary Pyrene compounds* λ_(abs)/λ_(cm) (nm) Dye No.Structure (H₂O) 44

45

46

400/424 For simplicity, any counter ions for the structures are notshown.

The present invention provides a method of preparing a compound of theinvention, the method comprising the steps of: 1) reacting a compoundcomprising a fluorophore linked to an amine-reactive group with an aminocompound having the following structural formula:

wherein n may be an integer selected from about 3 to about 30; h is aninteger selected from 1 to 5; and R_(p) is a latent or a protectedreactive group; 2) converting the latent or protected reactive groupR_(p) in the resulting compound to a reactive group.

The above amino compound shown above can be readily prepared, forexample, by reacting a suitable mPEG alkylating compound, such as a mPEGtosylate or mPEG chloride, with a suitable aminoalkyl compoundcomprising a suitable R_(p) group. Suitable R_(p) groups include acarboxylic acid group, a methyl ester of a carboxylic acid group, at-butyl ester group of a carboxylic acid group, a t-BOC-protected aminegroup and a benzyloxycarbonyl-protected group, merely by way of example.If the R_(p) group is a latent reactive group, such as a free carboxylicacid group, it may be directly converted to a reactive group usingmethods well known to one of skills. If R_(p) is a protected group, itis deprotected and then converted to a reactive group using a knownmethod.

(1) Uses of the Subject Compounds.

The subject compounds find use in a variety of different applications.One application of interest is the use of the subject compounds aslabeling agents which are capable of imparting a fluorescent property toa particular composition of matter. The compounds of the presentinvention can be used to react with any of a broad range of molecules,including but not limited to, biomolecules such as polypeptides,polypeptide-based toxins, amino acids, nucleotides, polynucleotidesincluding DNA and RNA, lipids, and carbohydrates, and any combinationsthereof. Additionally, the compounds of the invention can be used toreact with haptens, drugs, ion-complexing agents such as metalchelators, microparticles, synthetic or natural polymers, cells,viruses, other fluorescent molecules including the dye moleculeaccording to the invention, or surfaces. The substrate moleculestypically comprise one or more functional groups, which react with thereactive group of the subject compounds to form covalent or non-covalentlinkage. In one aspect, the reactive group of a compound of theinvention is an activated ester (such as a succinimidyl ester, or SE), amaleimide, a hydrazide or an aminooxy group. Accordingly, in someaspects, functional group from a substrate molecule (or reactionsubstrate) is an amine, a thiol, an aldehyde or ketone. The resultingfluorescently labeled substrate molecules may be referred to asconjugates or labeled substrate molecules. Any methods practiced in theart (e.g., Brinkley, Bioconjugate Chem. 3, 2 (1992), incorporated hereinby reference) for preparing fluorescent group-substrate conjugates areapplicable for practicing the subject invention.

Conjugates of biomolecules and compounds of the invention usually havehigh fluorescence yield while typically retaining the criticalparameters of unlabeled biomolecules, such as solubility, selectivebinding to a receptor or nucleic acid, activation or inhibition of aparticular enzyme or the ability to incorporate into a biologicalmembrane. Nevertheless, conjugates with the highest degree of labelingmay still precipitate or bind nonspecifically. As necessary, aless-than-maximal degree of labeling may be acceptable in order topreserve function or binding specificity. Preparing the conjugates ofthe invention may involve experimentation to optimize properties.Following conjugation, unconjugated labeling reagent may be removed bytechniques known in the art such as by gel filtration, dialysis,conjugate precipitation and resolubilization, HPLC or a combination ofthese techniques. The presence of free dye, particularly if it remainschemically reactive, may complicate subsequent experiments with thebioconjugate.

Nucleic Acids

In another embodiment, the subject compounds can be used to conjugatewith a nucleoside, a nucleotide, or a polynucleotide, wherein any ofsuch molecules may be natural or synthetic, modified or unmodified. Thecompound of the invention used for labeling may comprise a reactivegroup which is a phosphoramidite, an activated ester (such as asuccinimidyl ester), an alkylating group or a reactive platinum complex.Such molecules may contain or are derivatized to contain one or morereaction partners for the reactive groups on the compounds of theinvention. A reactive group of a compound of the invention may reactwith a suitable reaction partner on said molecule to form a covalentlinkage. For example, a phosphoramidite group may react with a hydroxylgroup to form a phosphate linkage after deprotection; a succinimidylester or the like may react with an amine group to form an amidelinkage; and a reactive platinum complex may react with a guanosine baseto form a platinum complex linkage. In one embodiment, a reactivecompound of the invention comprising an activated ester is reacted witha nucleotide triphosphate comprising a base comprising an aminoalkynylgroup, an aminoallyl group or an aminoalkyl group to form afluorescently labeled nucleotide triphosphate. Such a labeled nucleotidetriphosphate is often used to prepare a fluorescently labeled nucleicacid polymer via enzymatic incorporation.

In some embodiments, the fluorescent compound of the invention isreacted with a group or linker attached to the C-5 position of a uridineor cytidine residue. This position is not involved in Watson-Crickbase-pairing and interferes little with hybridization to complementarysequences. An aminoalkynyl linker may be introduced between afluorescent moiety and the nucleotide in order to reduce fluorophoreinteraction with enzymes or target binding sites. In addition to thisfour-atom bridge, seven- to 10-atom spacers may be introduced thatfurther separate the fluorophore from the base. The use of longerspacers may result in brighter conjugates and increased haptenaccessibility for secondary detection reagents.

Alternatively, deoxycytidine triphosphates may be prepared which aremodified at the N-4 position of cytosine using a 2-aminoethoxyethyl(OBEA) linker. Possible steric interference caused by the presence ofthe fluorescent fluorophore may be reduced by the use of additionalspacers.

Fluorescently labeled DNA may be prepared from a fluorescently labelednucleotide triphosphate by PCR reaction, terminal transferase-catalyzedaddition or nick translation. Various polymerases may be used in suchreactions. Such polymerases include Taq polymerase (useful e.g. inpolymerase chain reaction (PCR) assays), DNA polymerase I (useful e.g.in nick-translation and primer-extension assays), Klenow polymerase(useful e.g. in random-primer labeling), Terminal deoxynucleotidyltransferase (TdT) (useful e.g. for 3′-end labeling), Reversetranscriptase (e.g. for synthesizing DNA from RNA templates) or otherpolymerases such as SP6 RNA polymerase, T3 RNA polymerase and T7 RNApolymerase for in vitro transcription.

Alternatively, a fluorescently labeled nucleic acid polymer may beprepared by first enzymatically incorporating an amine-labelednucleotide into a nucleic acid polymer to result in an amine-labelednucleic acid polymer, followed by the labeling of said amine-labeledpolymer with a compound of the invention. More information on thepreparation and use of fluorescently labeled nucleotide triphosphatescan be found in U.S. Pat. Nos. 4,711,955 and 5,047,519. Stillalternatively, a nucleic acid polymer, such as a DNA, may be directlylabeled with a compound of the invention comprising a reactive platinumcomplex as the reactive group, wherein the platinum complex form acoordinative bond with a nitrogen atom of a guanosine base such asdescribed in U.S. Pat. No. 5,714,327.

Aminoacids and Polypeptides

In another embodiment, the subject compounds can be used to conjugatewith an amino acid, amino acid analog or a polypeptide. Labeledaminoacids, amino acid analogs and polypeptides may be labeled byreacting the compounds of the invention with amino acids, amino acidanalogs and polypeptides comprising reaction partners for the reactivegroups on said compounds. Such reaction partners may be natural orunnatural groups present in said polypeptides. By way of example,reaction partners may be the natural residues such as amino groups,which are part of natural lysine residues, or thiol groups, which arepart of natural cysteine groups.

In order to achieve the maximal fluorescence possible, a protein may belabeled with as many molecules of the same fluorescent group aspossible, to the degree that the biological activity of the protein isminimally affected by the labeling. In other cases it may be desirableto avoid fluorescence quenching resulting from multiple fluorescentgroup molecules on the protein interacting with each other. Dye-dyeinteractions may be physical, such as dye aggregation, or may be aspectral, such as FRET-based energy transfer, or a combination of both.Either type of interaction may lead to fluorescence quenching, which canbe characterized by a slow rise and then a rapid drop of the totalfluorescence of the labeled protein as the degree of labeling increases.FIGS. 1-9 show that a fluorescent group of the invention that is lesslikely or substantially less likely to quench its fluorescence on anantibody or streptavidin than a similar fluorescent group of prior artwithout a water soluble polymer group. A primary reason for fluorescencequenching of a labeling fluorescent group on protein is believed to bedue to formation of dye aggregates such as dye dimer. When dye dimerformation occurs, the absorption spectrum of the fluorescentgroup-protein conjugate typically show a doublet peak. As shown in FIG.1, a fluorescent group of the invention is much less likely to havedimer formation than a similar fluorescent group of prior art.Consequently, the fluorescent group of the invention is much brighter onproteins (FIG. 2). The relatively low tendency to aggregate on proteinsfor fluorescent groups of the invention permits a protein to be labeledmultiple times (i.e., a higher degree of labeling (DOL)) for a greaterfluorescence intensity (FIGS. 3-8). As a result, antibodies labeled witha fluorescent group of the invention are more sensitive in detectingtheir targets than antibodies labeled with a fluorescent group of priorart (FIG. 9). Another advantage for antibodies labeled with afluorescent group of the invention is their improved stainingspecificity relative to fluorescently labeled antibodies (FIG. 10),e.g., antibodies labeled with a fluorescent group of the inventionretain higher binding specificity with their antigen than the sameantibody labeled with the same degree of labeling with a conventionalfluorescent dye. A further advantage of the fluorescent groups of theinvention is a consequence of decreased aggregation and increasedbinding specificity; a labeled biomolecule of the invention, uponbinding with its binding partner, will provide a fluorescent signal witha higher signal-to-noise ratio than the complex formed with the samebinding partner and the same biomolecule which has been labeled with aconventional fluorescent group that is not a compound of the invention.

In some embodiments, the complexes of the methods of the invention havea signal-to-noise ratio that is equal or greater than about 70, about80, about 90, about 100, about 110, about 120, about 130, about 140,about 150, about 160, about 170, about 180, about 190, about 200, about210, about 220, about 230, about 240, about 250, about 255, about 260,about 265, about 270, about 275, about 280, about 285, about 290, about295, about 300, about 305, about 310, about 315, about 320, about 330,about 340, about 350, about 360, about 370, about 380, about 390, orabout 400. In some embodiments of the complexes of the methods of theinvention, the signal-to-noise ratio is no less than about 100, about110, about 120, about 130, about 140, about 150, about 160, about 170,about 180, about 190, about 200, about 210, about 220, about 230, about240, about 250, about 255, about 260, about 265, about 270, about 275,about 280, about 285, about 290, about 295, about 300, about 305, about310, about 315, about 320, about 330, about 340, about 350, about 360,about 370, about 380, about 390, or about 400.

In some embodiments, a complex of a labeled biomolecule comprising alabel of the invention, upon binding with its binding partner, willprovide a fluorescent signal which is greater than that of a complexformed with the same binding partner and the same biomolecule which hasbeen labeled to the same degree of labeling with a conventionalfluorescent group by about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 80%, about 90%, about 100%, about 125%,about 150%, about 175%, about 200%, about 300%, about 400%, about 500%,about 600%, about 700%, about 800%, about 900%, or about 1000%.

Still another advantage of the fluorescent group is their highphotostability, which is of particular importance for fluorescencemicroscopy studies (FIG. 11). Additionally, a polypeptide labeled with afluorescent compound of the invention may have a serum half life noshorter than than of the corresponding polypeptide that has nofluorescent label.

(2) Uses of the Labeled Biomolecules of the Invention

The subject compounds provide an effective tool for labelingbiomolecules for a wide variety of applications. Labeling allows one todiscern interactions involving biomolecules such as proteins,glycoproteins, nucleic acids, and lipids, as well as inorganicchemicals, or any combinations thereof. The interactions may be betweennucleic acid molecules, between nucleic acid and protein, and betweenprotein and small molecules. The interactions may be discerned in acell-free biological system, in a cellular system (includingintracellular and extracellular systems), or in vivo, which encompasseswhich encompasses activities within a cell that is within a tissue ororgan or a subject Delineating the various interactions is often asignificant step in scientific research and development, drug design,screening and optimization, phylogenetic classification, genotypingindividuals, parental and forensic identification, environmentalstudies, diagnosis, prognosis, and/or treatment of disease conditions.

Biomolecules labeled according to the methods of the invention may beused as binding agents to detect their binding partners, the targets oftheir biological interaction, as described above. For example, a proteincan be labeled with a dye of the invention and used to bind to a cellsurface receptor. In some embodiments of the invention, a binding agentis labeled with a substituted cyanine dye having maximal fluorescenceexcitation wavelength of equal or greater than 660 nm, a water solublepolymer group, and a reactive group under conditions effective tocrosslink the dye and the binding agent. In some embodiments, thesubstituted cyanine dye is substituted by a non-spiro substituent. Abinding agent so labeled is contacted with its binding partner, and thefluorescent label is detected. In other embodiments, a binding agent isreacted with a compound of structure of Formula I, II, III, IV or Vunder conditions effective to crosslink the compound with the bindingagent

Labeled molecules of the invention may be used as part of FRET pairs ina variety of biological assays and methods, whether as donor or acceptormolecules. A person skilled in the art will know to select a suitableFRET partner based on the specific application. Such applicationsinclude, but are not limited to, assays involving molecular beacons,FRET protease assays, flow cytometry, nucleic acid hybridization and anyother applications where the relative spatial localization of two ormore moieties must be probed. FRET is generally useful on scales of 10to 100 {acute over (Å)}. In one embodiment, both the donor and theacceptor of a FRET pair are labeled molecules of the invention. Inanother embodiment, one member of a FRET pair is a labeledoligonucleotide of the invention which is capable of annealing to acomplementary oligonucleotide labeled with a second member of the FRETpair, such that annealing leads to an increase in the efficiency ofenergy transfer. In this example, the second member of the FRET pair maybe a fluorophore of the invention or may be a different fluorophore.

In some applications, it is desirable to quench the labeled molecules ofthe invention. A variety of quenchers known in the art may be used.Non-limiting examples include Black Hole Quencher™ moieties, DABCYL,Reactive Red 4 (Cibacron Brilliant Red 3B-A), Malachite Green,4-Dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), and4,4′-Diisothiocyanaitodihydro-stilbene-2,2′-disulfonic acid. By way ofexample, a molecular beacon may be labeled with a compound of theinvention as well as with a suitable quencher. In the closedconformation of the beacon, the fluorophore is quenched. When the beaconopens as a result of a recognition or binding event, the fluorescence ofthe fluorophore increases significantly.

In still another embodiment, the invention provides an energy transferfluorescent group comprising a first donor fluorescent group and secondacceptor fluorescent group wherein: the donor fluorescent group andacceptor fluorescent group are covalently linked to form a FRET pair; atleast one of the donor fluorescent group and acceptor fluorescent groupis a fluorescent group of the invention; and the energy transferfluorescent group optionally comprises a reactive group. Methods forpreparing energy transfer fluorescent groups and uses thereof have beenpreviously described. See U.S. Pat. No. 6,479,303 and WO 00/13026.

In one embodiment, a fluorescent group of the invention is used to labela fluorescent protein to form a so-called tandem dye, wherein thefluorescent group of the invention and the fluorophore of thefluorescent protein form an energy transfer pair (i.e., FRET pair). Insuch a FRET pair, the fluorescent group of the invention is either thedonor fluorescent group or the acceptor fluorescent group and, likewise,the fluorophore of the protein is either the acceptor fluorescent groupor the donor fluorescent group, such that the FRET pair can be excitedat or near the absorption maxima of the donor fluorescent group and thefluorescence collected at the emission maxima of the acceptorfluorescent group, resulting in a large Stokes shift. Suitablefluorescent proteins for preparing tandem dyes include, but are notlimited to, various phycobiliproteins such as Allophycocyanin B,Allophycocyanin (APC), C-Phycocyanin, R-Phycocyanin, Phycoerythrocyanin,C-Phycoerythrin, b-Phycoerythrin, B-Phycoerythrin, R-Phycoerythrin(R-PE), and the likes. Phycobiliproteins are proteins comprising bilinas prosthetic groups, which are also the fluorophores of the proteins.Preferably, the phycobiliproteins are R-PE or APC. To achieve suitableFRET efficiency, one may choose a fluorescent group of properwavelengths so that the emission of the donor fluorescent group and theabsorption of the acceptor fluorescent group have sufficient spectraloverlap. Detailed methods for fluorescent group selection and forpreparing tandem dyes are disclosed in U.S. Pat. Nos. 4,520,110 and5,714,386. Because of their large Stokes shift, tandem dyes of theinvention may be useful for multi-color detections where only a limitednumber of excitation light sources may be available. In particular,tandem dyes of the invention may be useful for fluorescence-activatedcell sorting (FACS) or flow cytometry studies. Commercial flowcytometers are typically equipped with 1 to 3 excitation light sources,more commonly 1 to 2 excitation light sources. For example, some of thecommercial flow cytometers are equipped with a 488 nm argon laser and a633 nm He—Ne laser or a 635 nm red diode laser, and a significant numberof flow cytometers have only the 488 nm argon laser. Thus, in order todetect multiple targets, each target may be stained with a differentfluorescent group having a different emission and the differentfluorescent groups all need to be efficiently excited by a commonexcitation source. Tandem dyes of the invention can fill this need asdifferent tandem dyes having the same excitation maxima but differentemission maxima can be readily prepared. For example, R-PE may belabeled with Dye No. 10 of Table 3 and compound No. 18 of Table 3,respectively, to result in two tandem dyes where the first tandem dye isexcitable at 488 nm with emission at 665 nm and the second tandem dye isalso excitable at 488 nm but with emission at 775 nm.

In one embodiment, a compound of the invention is applied to abiological sample comprising a plurality of polypeptides and optionallyother biological molecules under a condition facilitating the covalentlabeling of said polypeptides. In some embodiments, the reactive groupof the compound is an activated ester, a maleimide, an iodoacetamide, abromoacetamide, a hydrazide, an amine or an aminooxy group. Thebiological sample may be a cell lysate or a tissue lysate. The resultinglabeled polypeptides or cellular components may be analyzed and/orpurified by any of a variety of known tools or techniques, including,but not limited to, protein microarrays, chromatography and gelelectrophoresis.

The present invention also provides kits comprising compounds of theinvention and/or fluorescent group-substrate conjugates of the inventionfor various assays as selectively described above. A kit of theinvention may comprise one or more compounds of the invention andinstructions instructing the use of said compound. For example, a kitmay comprise one or more compounds of the invention for labeling asubstrate, one or more buffers for the labeling reaction and productpurification, a chromatography column for purifying the resultingfluorescent group-substrate conjugate, a protocol for carrying out theprocedure, optionally any additional reagents and optionally anyreference standard. In another embodiment, a kit comprises one or morefluorescent group-substrate conjugates of the invention, one or morebuffers, a protocol for the use of said conjugate(s), optionally anyother reagents for an assay, and optionally any calibration standard(s).The kit may further contain other materials or devices of use inpurifying the conjugation products.

The signals produced by the fluorescent groups of the invention may bedetected in a variety of ways. Generally, a change of signal intensitycan be detected by any methods known in the art and is generallydependent on the choice of fluorescent group used. It can be performedwith the aid of an optical system. Such system typically comprises atleast two elements, namely an excitation source and a photon detector.Numerous examples of these elements are available in the art. Anexemplary excitation source is a laser, such as a polarized laser. Thechoice of laser light will depend on the fluorescent group attached tothe probe. For most of the fluorescent groups, the required excitationlight is within the range of about 300 nm to about 1200 nm, or morecommonly from about 350 nm to about 900 nm. Alternatively, compounds ofthe invention may be excited using an excitation wavelength of about 300to about 350 nm, 350 to 400 nm, 400 to 450 nm, 450 to 500 nm, 500 to 550nm, 550 to 600 nm, 600 to 650 nm, 650 to 700 nm, 750 nm to 800 nm, orfrom 800 nm to 850 nm, merely by way of example. Those skilled in theart can readily ascertain the appropriate excitation wavelength toexcite a given fluorophore by routine experimentation (see e.g., TheHandbook—‘A Guide to Fluorescent Probes and Labeling Technologies, TenthEdition’ (2005) (available from Invitrogen, Inc./Molecular Probes)previously incorporated herein by reference). Where desired, one canemploy other optical systems. These optical systems may compriseelements such as optical reader, high-efficiency photon detectionsystem, photo multiplier tube, gate sensitive FET's, nano-tube FET's,photodiode (e.g. avalanche photo diodes (APD)), camera, charge coupledevice (CCD), electron-multiplying charge-coupled device (EMCCD),intensified charge coupled device (ICCD), and confocal microscope. Theseoptical systems may also comprise optical transmission elements such asoptic fibers, optical switches, mirrors, lenses (including microlens andnanolens), collimators. Other examples include optical attenuators,polarization filters (e.g., dichroic filter), wavelength filters(low-pass, band-pass, or high-pass), wave-plates, and delay lines. Insome embodiments, the optical transmission element can be planarwaveguides in optical communication with the arrayed opticalconfinements. See, e.g., U.S. Pat. Nos. 7,292,742, 7,181,122, 7,013,054,6,917,726, 7,267,673, and 7,170,050. These and other optical componentsknown in the art can be combined and assembled in a variety of ways toeffect detection of distinguishable signals.

Fluorescently labeled polynucleotides of the invention find use in avariety of applications. Such applications can involve interactionsbetween nucleic acids, e.g., interactions between DNA and DNA, DNA andRNA, and RNA and RNA, or any other non-naturally occurring nucleic acidsPNA, LNA, and/or TNA. Various applications can also involve interactionsbetween nucleic acids and proteins, lipids or combinations thereof.Non-limiting examples of specific nucleic acid assays include nucleicacid amplification, both quantitative or end-point amplification,hybridization in solution or on a substrate (e.g., array hybridization),gel shifts, and nucleic acid sequencing. The fluorescently labeledpolynucleotides can be used in solution phase or immobilized on asubstrate.

In one embodiment, the labeled polynucleotides are used as hybridizationprobes. One application of hybridization probes is fluorescent in situhybridization (FISH). In this technique, a labeled polynucleotidecomplementary to a sequence of interest is annealed to fixed chromosomespreparations, and the presence of the sequence of interest as well asthe chromosomal localization is detected by microscopy. FISH can beperformed by immobilizing the nucleic acids of interest on a substrateincluding without limitation glass, silicon, or fiber. FISH may also beused quantitatively (Q-FISH) to detect the presence and length ofrepetitive sequences such as telomeres. This may be done by quantitatingthe intensity of emitted fluorescence as measured by microscopy. FISHassays utilizing the subject fluorescent compounds can be performed fordetecting a specific segment of a DNA molecule or a chromosome. Thesefeatures can be used in genetic counseling (e.g., prenatal-screens),medicine, and species identification.

In some embodiments, labeled polynucleotides can be used as primers inamplification reactions such as PCR. In yet another embodiment, acompound of the invention may be used to label a polynucleotide which issubsequently used as a probe may be a hybridization probe or a real-timePCR probe. Such a probe may be labeled with a second fluorescent groupto form a FRET pair with the first fluorescent group of the invention.Methods for the preparation and use of PCR probes are well known to oneskilled in the art.

In one embodiment of the invention, a method is provided for detectingor quantifying a target nucleic acid, the method comprising the stepsof: a) providing a labeled polynucleotide (“probe”) of the presentinvention; b) contacting said labeled polynucleotide with the nucleicacid target so as to allow for hybridization of the probe with thenucleic acid target; and c) detecting or quantifying said nucleic acidtarget by measuring a change in the fluorescence of the probe upon thehybridization of the nucleic acid probe with the nucleic acid target.

As used herein, hybridization occurs when the probe form a complex withthe target nucleic acid. In general, the complex is stabilized, at leastin part, via hydrogen bonding between the bases of the nucleotideresidues. The hydrogen bonding may occur by Watson-Crick base pairing,Hoogstein binding, or in any other sequence-specific manner.Hybridization may constitute a step in a more extensive process, such asthe initiation of a PCR reaction, or the enzymatic cleavage of apolynucleotide by a ribozyme.

After hybridization between the probe and the target has occurred, achange in the intensity of the fluorescence of the probe may bemeasured. Such change before and after hybridization can yield apositive gain or negative reduction in the detected signal intensity.Depending on the specific hybridization assay that is run, more than oneevent after hybridization may contribute to the generation of a changein signal intensity. For example, an increase in reporter signal mayresult by way of spatial extension or separation of the reporterfluorescent group from the quencher group while both are still attachedto the probe. In addition, either the reporter or the quencher of theprobe can be separated by way of cleavage via an enzyme (e.g., apolymerase having a 5′ to 3′ exonuclease), thereby generating a reportersignal that is detected. As noted above, both the reporter and thequencher are defined in functional terms, such that these groups can beidentical though serving, relative to each other, a different functionwhen used in a hybridization reaction. For example, a group attached toa probe is a quencher because it reduces the emission of an opticalsignal when the probe is not hybridized with the target nucleic acid(typically when the probe assumes a random state). The same group canbecome a reporter fluorescent group upon being cleaved by an enzymeafter hybridization with the target nucleic acid as the signal of thefluorescent group is now detected during the assay.

The signal detection methods described previously can be applied tonucleic acid amplification in which the target nucleic acid is increasedin copy number. Such increase may occur in a linear or in an exponentialmanner. Amplification may be carried out by natural or recombinant DNApolymerases such as Taq polymerase, Pfu polymerase, T7 DNA polymerase,Klenow fragment of E. coli DNA polymerase, Tma DNA polymerase, exo-TliDNA polymerase, exo-KOD DNA polymerase, exo-JDF-3 DNA polymerase,exo-PGB-D DNA polymerase, U1Tma (N-truncated) Thermatoga martima DNApolymerase, Sequenase, and/or RNA polymerases such as reversetranscriptase.

A preferred amplification method is polymerase chain reaction (PCR).General procedures for PCR are taught in U.S. Pat. No. 4,683,195(Mullis) and U.S. Pat. No. 4,683,202 (Mullis et al.). Briefly,amplification of nucleic acids by PCR involves repeated cycles ofheat-denaturing the DNA, annealing two primers to sequences that flankthe target nucleic acid segment to be amplified, and extending theannealed primers with a polymerase. The primers hybridize to oppositestrands of the target nucleic acid and are oriented so that thesynthesis by the polymerase proceeds across the segment between theprimers, effectively doubling the amount of the target segment.Moreover, because the extension products are also complementary to andcapable of binding primers, each successive cycle essentially doublesthe amount of target nucleic acids synthesized in the previous cycle.This results in exponential accumulation of the specific target nucleicacids at approximately a rate of 2^(n), where n is the number of cycles.

A typical conventional PCR thermal cycling protocol comprises 30 cyclesof (a) denaturation at a range of 90° C. to 95° C. for 0.5 to 1 minute,(b) annealing at a temperature ranging from 50° C. to 65° C. for 1 to 2minutes, and (c) extension at 68° C. to 75° C. for at least 1 minute.Other protocols including but not limited to universal protocol as wellas fast cycling protocol can be performed the subject probes as well.

A variant of the conventional PCR is a reaction termed “Hot Start PCR”.Hot Start PCR techniques focus on the inhibition of polymerase activityduring reaction preparation. By limiting polymerase activity prior toPCR cycling, non-specific amplification is reduced and the target yieldis increased. Common methods for Hot Start PCR include chemicalmodifications to the polymerase (see, e.g., U.S. Pat. No. 5,773,258),inhibition of the polymerase by a polymerase-specific antibody (see,e.g., U.S. Pat. No. 5,338,671), and introduction of physical barriers inthe reaction site to sequester the polymerase before the thermal cyclingtakes place (e.g., wax-barrier methods). The reagents necessary forperforming Hot Start PCR are conveniently packaged in kits that arecommercially available (see, e.g., Sigma's JumpStart Kit).

Another variation of the conventional PCR that can be performed with thesubject probes is “nested PCR” using nested primers. The method ispreferred when the amount of target nucleic acid in a sample isextremely limited for example, where archival, forensic samples areused. In performing nested PCR, the nucleic acid is first amplified withan outer set of primers capable of hybridizing to the sequences flankinga larger segment of the target nucleic acid. This amplification reactionis followed by a second round of amplification cycles using an inner setof primers that hybridizes to target sequences within the large segment.

The subject probes can be employed in reverse transcription PCR reaction(RT-PCR), in which a reverse transcriptase first coverts RNA moleculesto double stranded cDNA molecules, which are then employed as thetemplate for subsequent amplification in the polymerase chain reaction.In carrying out RT-PCR, the reverse transcriptase is generally added tothe reaction sample after the target nucleic acids are heat denatured.The reaction is then maintained at a suitable temperature (e.g., 30°C.-45° C.) for a sufficient amount of time (e.g., 5-60 minutes) togenerate the cDNA template before the scheduled cycles of amplificationtake place. Such reaction is particularly useful for detecting thebiological entity whose genetic information is stored in RNA molecules.Non-limiting examples of this category of biological entities includeRNA viruses such as HIV and hepatitis-causing viruses. Another importantapplication of RT-PCR embodied by the present invention is thesimultaneous quantification of biological entities based on the mRNAlevel detected in the test sample.

The subject probes can also be employed to perform ligase chainpolymerase chain reaction (LCR-PCR). The method involves ligating thetarget nucleic acids to a set of primer pairs, each having atarget-specific portion and a short anchor sequence unrelated to thetarget sequences. A second set of primers containing the anchor sequenceis then used to amplify the target sequences linked with the first setof primers. Procedures for conducting LCR-PCR are well known to artisansin the field, and hence are not detailed herein (see, e.g., U.S. Pat.No. 5,494,810).

The subject probes are particularly suited for use in a homogeneousassay. In such an assay, a target nucleic acid is detected and/orquantified without the requirement of post-assay processing to recordthe result of the assay. For example, a homogeneous PCR reaction can becarried out in a closed sample holder (e.g., a tube, a sample capillaryor thermalchip), and no further addition or removal of reagents isnecessary to record the result once the assay is started. Homogeneousassays allow recordation of the result of the assay in real time. Wheredesired, in practicing the subject methods, the result of the assay canbe continuously recorded as the assay progresses in time or recordedintermittently at one or more point during the assay or upon completionof the assay.

Where desired, homogeneous assays can be multiplexed, i.e., more thanone target nucleic acid can be detected in one assay. In a multiplexassay, two or more specific nucleic acid probes, which differ in thenature of their covalently attached fluorescent groups, are added to themixture to be assayed. The fluorescent groups are chosen to producedistinguishable fluorescent signals from each specific nucleic acidprobe. The signals of the different fluorescent group combinations ofthe nucleic acid probes can be recorded simultaneously to detect and/orquantify the corresponding target nucleic acids. Multiplexing greatlyreduces the cost of analysis and can tremendously increase throughput inhigh volume settings.

The subject probes can be used to detect single mutations. Accordingly,methods are provided to use the probes of the invention to detect as fewas a single mismatch between the probe sequence and a target sequence.Such high specificity in nucleic acid detection by PCR is highlyvaluable in clinical diagnosis and genetic research. For example, manydiseases are associated with single mutations at different sites in thehuman genome. Although in theory this type of genetic variations, alsocalled single nucleotide polymorphism or SNP, may be detected bysequencing, such sequencing method is not expected to be practical on alarge scale due to high cost and low efficiency. Detection of SNP by anamplification reaction is feasible with the use of the subject probes.

The subject probes are also particularly suited for monitoring nucleicacid amplification reactions. In a related embodiment, the presentinvention provides a method of monitoring the increase in a targetnucleic acid during amplification of said target. The method typicallyinvolves a) providing an amplification reaction mixture that comprisessaid target nucleic acid, at least one primer that hybridizes to thetarget nucleic acid, a labeled oligonucleotide probe of the presentinvention that provides a detectable signal, the intensity of which isproportional to the increase in the target nucleic acid in theamplification; (b) treating said mixture under conditions for amplifyingsaid target nucleic acid; and (c) measuring the amount of said signalproduced by said mixture during said treating step (c). Where desired,the amount of signal is determined continuously throughout theamplification reaction or determined intermittently during theamplification reaction. The amplification can be exponentially with theuse of a primer pair or linearly with the use of one primer of the pair.

The increase in signal intensity during the amplification reaction maydue to the step of hybridization of the probe to the target nucleic acidand also the step of cleavage via the action of the polymerase utilizedin the amplification reaction.

In one aspect, the subject methods exploit the 5′ to 3′ nucleaseactivity of a polymerase when used in conjunction with PCR. When thesubject probe is added concomitantly with the primer at the start ofPCR, and the signal generated from hydrolysis of the labelednucleotide(s) of the probe provides a means for detection of the targetsequence during its amplification. Numerous polymerases are suited tocatalyze primer and template-dependent nucleic acid synthesis andpossess the 5′ to 3′ nuclease activity. Non-limiting examples includeDNA polymerases such as E. coli DNA polymerase I, Thermus thermophilus(Tth) DNA polymerase, Bacillus stearothermophilus DNA polymerase,Thermococcus littoralis DNA polymerase, and Thermus aquaticus (Taq) DNApolymerase. Where desired, temperature stable polymerases can beemployed in a nucleic acid amplification reaction. See, e.g., U.S. Pat.No. 4,889,818 that discloses a representative thermostable enzymeisolated from Thermus aquaticus. Additional representative temperaturestable polymerases include without limitation, e.g., polymerasesextracted from the thermostable bacteria Thermus flavus, Thermus ruber,Thermus thermophilus, Bacillus stearothermophilus (which has a somewhatlower temperature optimum than the others listed), Thermus lacteus,Thermus rubens, Thermotoga maritima, Thermococcus littoralis, andMethanothermus fervidus.

In another embodiment, nucleic acid amplification can be performed withpolymerases that exhibit strand-displacement activity (also known asrolling circle polymerization). Strand displacement can result in thesynthesis of tandem copies of a circular DNA template, and isparticularly useful in isothermal PCR reaction. Non-limiting examples ofrolling circle polymerases suitable for the present invention includebut are not limited to T5 DNA polymerase (Chatterjee et al., Gene97:13-19 (1991)), and T4 DNA polymerase holoenzyme (Kaboord andBenkovic, Curr. Biol. 5:149-157 (1995)), phage M2 DNA polymerase(Matsumoto et al., Gene 84:247 (1989)), phage PRD1 DNA polymerase (Junget al., Proc. Natl. Aced. Sci. USA 84:8287 (1987), and Zhu and Ito,Biochim. Biophys. Acta. 1219:267-276 (1994)), Klenow fragment of DNApolymerase I (Jacobsen et al., Eur. J. Biochem. 45:623-627 (1974)).

A preferred class of rolling circle polymerases utilizes protein primingas a way of initiating replication. Exemplary polymerases of this classare modified and unmodified DNA polymerase, chosen or derived from thephages Φ29, PRD1, Cp-1, Cp-5, Cp-7, Φ15, Φ1, Φ21, Φ25, BS 32 L17, PZE,PZA, Nf, M2Y (or M2), PR4, PR5, PR722, B103, SF5, GA-1, and relatedmembers of the Podoviridae family. Specifically, the wildtypebacteriophage Φ29 genome consists of a linear double-stranded DNA(dsDNA) of 19,285 base pairs, having a terminal protein (TP) covalentlylinked to each 5′ end. To initiate replication, a histone-like viralprotein forms a nucleoprotein complex with the origins of replicationthat likely contributes to the unwinding of the double helix at both DNAends (Serrano et al., The EMBO Journal 16(9): 2519-2527 (1997)). The DNApolymerase catalyses the addition of the first dAMP to the hydroxylgroup provided by the TP. This protein-primed event occurs opposite tothe second 3′ nucleotide of the template, and the initiation product(TP-dAMP) slides back one position in the DNA to recover the terminalnucleotide After initiation, the same DNA polymerase replicates one ofthe DNA strands while displacing the other. The high processivity andstrand displacement ability of Φ29 DNA polymerase makes it possible tocomplete replication of the Φ29 TP-containing genome (TP-DNA) in theabsence of any helicase or accessory processivity factors (reviewed bySerrano et al, The EMBO Journal 16(9): 2519-2527 (1997)).

Strand displacement can be enhanced through the use of a variety ofaccessory proteins. They include but are not limited to helicases(Siegel et al., J. BioL Chem. 267:13629-13635 (1992)), herpes simplexviral protein ICP8 (Skaliter and Lehman, Proc. Natl, Acad. Sci. USA91(22):10665-10669 (1994)), single-stranded DNA binding proteins (Riglerand Romano, J. Biol. Chem. 270:8910-8919 (1995)), adenovirus DNA-bindingprotein (Zijderveld and van der Vliet, J. Virology 68(2):1158-1164(1994)), and BMRF1 polymerase accessory subunit (Tsurumi et al., J.Virology 67(12):7648-7653 (1993)).

The subject probes can be utilized in an isothermal amplificationreaction. Such amplification reaction does not rely solely upon thermalcycling. The procedure can be applied at a wide range of ambienttemperatures. In particular, denaturation of the double-strandedtemplate sequence is not accomplished solely through an increase intemperature above the melting temperature of the double strandedsequence. Rather, the denaturation process involves physical ormechanical force that separates the strand to allow primer annealing andextension. Various mechanisms for conducting isothermal amplificationreaction including isothermal PCR are described in US. PatentPublication No 20060019274 and U.S. Pat. Nos. 5,824,477 and 6,033,850,which are incorporated herein by reference.

Nucleic acid amplification is generally performed with the use ofamplification reagents. Amplification reagents typically includeenzymes, aqueous buffers, salts, primers, target nucleic acid, andnucleoside triphosphates. Depending upon the context, amplificationreagents can be either a complete or incomplete amplification reactionmixture.

The choice of primers for use in nucleic acid amplification will dependon the target nucleic acid sequence. Primers used in the presentinvention are generally oligonucleotides, e.g., 10 to 100 or 10 to 25bases in length, that can be extended in a template-specific manner viathe action of a polymerase. In general, the following factors areconsidered in primer design: a) each individual primer of a pairpreferably does not self-hybridize in an amplification reaction; b) theindividual pairs preferably do not cross-hybridize in an amplificationreaction; and c) the selected pair must have the appropriate length andsequence homology in order to anneal to two distinct regions flankingthe nucleic acid segment to be amplified. However, not every nucleotideof the primer must anneal to the template for extension to occur. Theprimer sequence need not reflect the exact sequence of the targetnucleic acid. For example, a non-complementary nucleotide fragment maybe attached to the 5′ end of the primer with the remainder of the primersequence being complementary to the target. Alternatively,non-complementary bases can be interspersed into the primer, providedthat the primer sequence has sufficient complementarily with the targetfor annealing to occur and allow synthesis of a complementary nucleicacid strand.

A nucleic acid amplification reaction typically comprises a targetnucleic acid in a buffer compatible with the enzymes used to amplify thetarget. The buffer typically contains nucleotides or nucleotide analogs(ATP, TTP, CTP, GTP, or analogs thereof including without limitationpentaphosphates having the respective base unit) that are capable ofbeing incorporated into a replica strand of the template sequence.

Where desired, amplification reaction is carried out as an automatedprocess. Numerous thermocyclers are available in the art that arecapable of holding 48, 96 or more samples. A suitable optical systemmoves the excitation light from the source to the reaction sites andmeasures the emission light from each sample. For example, multiplefiber optic leads simultaneously read all PCR tubes undergoingthermocycling. However, only a single fluorometer may be needed to readfluorescence from the reaction sites. An analogous detection scheme issuitable in a 96-well microtiter format. This type of format isfrequently desirable in clinical laboratories for large scale samplescreening, for example, for genetic analysis such as screening for AIDSvirus in blood bank screening procedures.

Accordingly, the present invention also provides an apparatus fordetecting the signal generated by the subject probe, which can be usedto detect, measure, and quantify the signal before, during, and afteramplification. The apparatus comprises a thermal unit (e.g., athermocycler) capable of holding an amplification reaction mixturecomprising the subject probes and effecting an amplification of thetarget sequence, and a detector that detects the signal generated fromthe subject probes.

In another embodiment of the present invention, the subject probes areemployed in assays that are conducted on nucleic acid microarrays todetect or quantify nucleic acid targets. In such assays, a fluorescentsignal is generated on a nucleic acid microarray upon the presence of acomplementary target nucleic acid.

Nucleic acid microarrays including gene chips comprise ordered arrays ofnucleic acids that are covalently attached to a solid surface, see e.g.,U.S. Pat. Nos. 5,871,928, 6,040,193, 6,262,776, 6,403,320, and6,576,424. The fluorescent signal that is generated in the assay can bemonitored and quantified with optical detectors including but notlimited to fluorescence imagers, e.g. commercial instruments supplied byHitachi Corp., San Bruno, Calif. or confocal laser microscopes (confocalfluorescence scanners), e.g. commercial instruments from GeneralScanning, Inc., Watertown, Mass.

In assays that are conducted on nucleic acid microarrays, the targetnucleic acids may be provided as a mixture of nucleic acid sequencesderived from any suitable biological sources. They can be derived frombody fluid, solid tissue samples, tissue cultures or cells derivedtherefrom and the progeny thereof, and sections or smears prepared fromany of these sources, or any other samples that contain nucleic acids.

Where expression pattern is assayed, the mRNA sequences are firsttypically amplified by reverse transcription PCR with universal primersprior to their use as the target sequences in the assay. In oneembodiment, all nucleic acid sequences present in the test sample aresimultaneously applied to the microarray for analysis, thus allowing theinteraction of all target nucleic acid sequences with all nucleic acidsthat are present on the array. In another embodiment, the target nucleicacids applied to the array are pre-selected to yield a subset forrefined hybridization analysis utilizing a microarray. For example, alimited number of target sequences can contain more than one stretch ofspecific nucleotide sequence to be analyzed, e.g. more than one singlenucleotide polymorphism. The nucleic acid sequences of this setting maybe amplified by PCR with the aid of specific primers prior to theiranalysis on the microarray.

In assaying for expression of multiples genes of a subject, targetpolynucleotides are allowed to form stable complexes with probes on theaforementioned arrays in a hybridization reaction. It will beappreciated by one of skill in the art that where antisense RNA is usedas the target nucleic acid, the sequence immobilized on the array arechosen to be complementary to sequences of the antisense nucleic acids.Conversely, where the target nucleic acid pool is a pool of sensenucleic acids, the sequence immobilized on the array are selected to becomplementary to sequences of the sense nucleic acids. Finally, wherethe nucleic acid pool is double stranded, the probes may be of eithersense and/or antisense as the target nucleic acids include both senseand antisense strands.

In one embodiment, labeled probes are utilized to perform a competitivehybridization on a microarray. In this assay format, a target nucleicacid from a test sample competes with a probe of the present inventionfor binding of a known sequence immobilized on the microarray. Theamount of labeled probes that will bind to the immobilized knownsequences is inversely proportional to the concentration ofcorresponding target nucleic acids in the test sample.

A variant hybridization assay involves the use of polymerases on amicroarray to enhance the signals of the probes by performing cleavageof the reporters. For example, a mixture of target sequences are firstallowed to hybridize with known sequences immobilized on the array.Unhybridized sequences are then washed away. Thereafter, probescorresponding to the target sequences are allowed to hybridize todifferent regions on the targets. Upon washing of the excessive unboundprobes, the reporter fluorescent groups on the hybridized probes arecleaved via the action of polymerases, thereby generating a detectablesignal that is indicative of the presence and/or quantity of a targetsequence initially present in the test sample.

Suitable hybridization conditions for use of the labeled probes of theinvention are such that the recognition interaction between the sequenceon the array and target is both sufficiently specific and sufficientlystable. As noted above, hybridization reactions can be performed underconditions of different “stringency”. Relevant conditions includetemperature, ionic strength, time of incubation, the presence ofadditional solutes in the reaction mixture such as formamide, and thewashing procedure. Higher stringency conditions are those conditions,such as higher temperature and lower sodium ion concentration, whichrequire higher minimum complementarity between hybridizing elements fora stable hybridization complex to form. Conditions that increase thestringency of a hybridization reaction are widely known and published inthe art. See, for example, (Sambrook, et al., (1989), supra).

In general, there is a tradeoff between hybridization specificity(stringency) and signal intensity. In a preferred embodiment, washingthe hybridized array prior to detecting the target-probe complexes isperformed to enhance the signal to noise ratio. Typically, thehybridized array is washed at successively higher stringency solutionsand signals are read between each wash. Analysis of the data sets thusproduced will reveal a wash stringency above which the hybridizationpattern is not appreciably altered and which provides adequate signalfor the particular polynucleotide probes of interest. Parametersgoverning the wash stringency are generally the same as those ofhybridization stringency. Other measures such as inclusion of blockingreagents (e.g. sperm DNA, detergent or other organic or inorganicsubstances) during hybridization can also reduce non-specific binding.

Imaging specific hybridization event on a microarray is typicallyperformed with the aid of an optical system. Non-limiting examples ofsuitable systems include camera, charge couple device (CCD),electron-multiplying charge-coupled device (EMCCD), intensified chargecoupled device (ICCD), and confocal microscope.

The microarray provides a positional localization of the sequence wherehybridization has taken place. The position of the hybridized regioncorrelates to the specific sequence, and hence the identity of thetarget expressed in the test sample. The detection methods also yieldquantitative measurement of the level of hybridization intensity at eachhybridized region, and thus a direct measurement of the level ofexpression of a given gene transcript. A collection of the dataindicating the regions of hybridization present on an array and theirrespective intensities constitutes a hybridization pattern that isrepresentative of a multiplicity of expressed gene transcripts of asubject. Any discrepancies detected in the hybridization patternsgenerated by hybridizing target polynucleotides derived from differentsubjects are indicative of differential expression of a multiplicity ofgene transcripts of these subjects.

In one aspect, the hybridization patterns to be compared can begenerated on the same array. In such case, different patterns aredistinguished by the distinct types of detectable labels. In a separateaspect, the hybridization patterns employed for the comparison aregenerated on different arrays, where discrepancies are indicative of adifferential expression of a particular gene in the subjects beingcompared.

The test nucleic acids for a comparative hybridization analysis can bederived from (a) cells from different organisms of the same species(e.g. cells derived from different humans); (b) cells derived from thesame organism but from different tissue types including normal ordisease tissues, embryonic or adult tissues; (c) cells at differentpoints in the cell-cycle; (d) cells treated with or without external orinternal stimuli. Thus, the comparative hybridization analysis using thearrays of the present invention can be employed to monitor geneexpression in a wide variety of contexts. Such analysis may be extendedto detecting differential expression of genes between diseased andnormal tissues, among different types of tissues and cells, amongstcells at different cell-cycle points or at different developmentalstages, and amongst cells that are subjected to various environmentalstimuli or lead drugs. Therefore, the expression detecting methods ofthis invention may be used in a wide variety of circumstances includingdetection of disease, identification and quantification of differentialgene expression between at least two samples, linking the differentiallyexpressed genes to a specific chromosomal location, and/or screening forcompositions that upregulate or downregulate the expression or alter thepattern of expression of particular genes.

The subject amplification and any other hybridization assays describedherein can be used to detect any target nucleic acids from any sourcessuspected to contain the target. It is not intended to be limited asregards to the source of the sample or the manner in which it is made.Generally, the test sample can be biological and/or environmentalsamples. Biological samples may be derived from human or other animals,body fluid, solid tissue samples, tissue cultures or cells derivedtherefrom and the progeny thereof, sections or smears prepared from anyof these sources, or any other samples that contain nucleic acids.Preferred biological samples are body fluids including but not limitedto urine, blood, cerebrospinal fluid, spinal fluid, sinovial fluid,semen, ammoniac fluid, cerebrospinal fluid (CSF), and saliva. Othertypes of biological sample may include food products and ingredientssuch as dairy items, vegetables, meat and meat by-products, and waste.Environmental samples are derived from environmental material includingbut not limited to soil, water, sewage, cosmetic, agricultural andindustrial samples, as well as samples obtained from food and dairyprocessing instruments, apparatus, equipment, disposable, andnon-disposable items.

Polynucleotides labeled according to the invention may also be used ingel shift assays. Such an assay, also known as electrophoretic mobilityshift assay (EMSA), gel mobility shift assay, band shift assay, or gelretardation assay, is a common technique used to study protein-DNA orprotein-RNA interactions. This procedure can determine if a protein ormixture of proteins is capable of binding to a given DNA or RNAsequence, and can sometimes indicate if more than one protein moleculeis involved in the binding complex. Labeled oligonucleotides may be usedin gel shift assays by performing electrophoresis and subsequentlydetermining the extent of migration of the labeled oligonucleotides inthe gel by visualizing the emission of the fluorescent label. Gel shiftassays may be performed in vitro concurrently with DNase footprinting,primer extension, and promoter-probe experiments when studyingtranscription initiation, DNA replication, DNA repair or RNA processingand maturation. Methods of performing gel shift assays are known. See,e.g. Garner, M. M. and Revzin, A. (1981) “A gel electrophoresis methodfor quantifying the binding of proteins to specific DNA regions:application to components of the Escherichia coli lactose operonregulatory system.” Nucleic Acids Res. 9:3047-3060 or Fried, M. andCrothers, D. M. (1981) “Equilibria and kinetics of lacrepressor-operator interactions by polyacrylamide gel electrophoresis.”Nucleic Acids Res., 9:6505-6525.

Fluorescently labeled polypeptides of the invention are useful in a widevariety of assays. Such assays can be performed to discern specificprotein-protein interactions, protein-nucleic acid interaction,interactions between a protein of interest and candidate inhibitors oractivators. Candidate inhibitors or activators include but are notlimited to antisense oligonucleotides, double stranded RNAs, ribozymes,a ribozyme derivatives, antibodies, liposomes, small molecules,inorganic or organic compounds. The subject assays can also be performedto study enzymatic kinetics, for e.g., drug design, screen and/oroptimization and can be performed using the fluorescently labeledpolypeptides in solution or immobilized on a solid substrate.

Of particular interest is a specific interaction between a cell surfacereceptor and its corresponding ligand. Cell surface receptors aremolecules anchored on or inserted into the cell plasma membrane. Theyconstitute a large family of proteins, glycoproteins, polysaccharidesand lipids, which serve not only as structural constituents of theplasma membrane, but also as regulatory elements governing a variety ofbiological functions. In another aspect, the specific protein-proteininteraction involves a cell surface receptor and an immunoliposome or animmunotoxin. In yet another aspect, the specific protein-proteininteraction may involve a cytosolic protein, a nuclear protein, achaperon protein, or proteins anchored on other intracellular membranousstructures. In yet another aspect, the specific protein-proteininteraction is between a target protein (e.g., an antigen) and anantibody specific for that antigen.

A specific interaction between a labeled polypeptide and an interactingentity is assayed by mixing the two entities under conditions suchinteraction is suspected to occur. Typically, the interaction isvisualized with the aid of an optical device. Where desired, theseentities can be placed within an optical confinement (see, e.g., U.S.Pat. Nos. 7,267,673, and 7,170,050). Where single molecule is to bedetected, each optical confinement contains only one target that isbeing investigated. This can be achieved by diluting a minute amount oftarget in a large volume of solution, such that deposition over an arrayof confinements results in a primary distribution, or a majority ofconfinements will have a single target molecule disposed there. Thelabeled polypeptide and the interacting entity can be immobilized ontothe inner surface of the optical confinement by any of the methodsavailable in the art. Such methods encompass the uses of covalent andnoncovalent attachments effected by a variety of binding moieties. Thechoice of the binding moieties will depend on the nature of the labeledpolypeptide and/or the interacting entity. One way to immobilize thelabeled polypeptide or the proteinaceous probe involves the use of thestreptavidin or avidin/biotin binding pair.

In one embodiment, the polypeptide to be reacted with a compound of theinvention comprises 3 to about 80 amino acids. Examples of suchpolypeptides include, but are not limited to, neuropeptides, cytokines,toxins and peptidase or protease substrates. Fluorescentlylabeled-neuropeptides, -cytokines and -toxins may be used to map orvisualize the distribution of the receptors specific to the respectivepeptides. □s an example, when labeled with a compound of the invention,phalloidin, which is a toxin with a cyclic peptide structure, can beused to stain F-actin filaments in cells. As another example, whenlabeled with a fluorescent group of the invention, □-bungarotoxin, apeptide-based snake toxin, can be used to detect acetylcholine receptor.Peptidase or protease substrates labeled with a fluorescent group of theinvention may be used to assay the activities of the peptidases orproteases, and used in screening drugs designed as inhibitors of thepeptidases or proteases. For example, a peptide comprising a peptidesequence cleavable by a peptidase may be labeled at one end of thepeptide sequence with a first fluorescent group, a fluorescence donorfluorescent group, selected from a fluorescent group of the inventionand at the other end of the peptide sequence with a second fluorescentgroup, a fluorescence acceptor fluorescent group (such as anotherfluorescent group from the invention or a quencher), where the first dyeand second dye form a fluorescence resonance energy transfer (FRET)pair. By detecting the fluorescence difference of either the donorfluorescent group or the acceptor fluorescent group of the FRET pairbefore and after the peptide is cleaved by said peptidase, the level ofenzyme activity can be assessed.

Other polypeptide conjugates that can be prepared according to theinvention include those of antibodies, lectins, enzymes, lipoproteins,albumins, avidin, streptavidin, annexins, protein A, protein G,transferrin, apotransferrin, phycobiliproteins and other fluorescentproteins, toxins, growth factors, tubulins, hormones, various receptorsand ion channels.

In one embodiment, compounds of the invention may be reacted withantibodies. Such antibodies may be primary or secondary depending on thedesired application. If the antigen to be detected is present in verysmall amounts, a secondary antibody may be used in order to providesignal amplification. Various secondary antibody isotypes may belabeled. Non-limiting examples of secondary antibody isotypes areAnti-mouse IgG, Anti-mouse IgM, Anti-rabbit IgG, Anti-rat IgG, Anti-ratIgM, Anti-guinea pig IgG, Anti-chicken IgG, Anti-hamster IgG, Anti-humanIgG, Anti-human IgM, Anti-goat IgG, Anti-mouse IgG, Anti-rabbit IgG,Anti-rat IgG, Anti-sheep IgG, Anti-goat IgG, Anti-mouse IgG, Anti-humanIgG, Anti-rat IgG, Anti-mouse IgG, Anti-human IgG, Anti-rat IgG,Anti-goat IgG, and Anti-rabbit IgG.

Alternatively, Fab fragments may be labeled with the compounds of theinvention. Such fragments may be superior to whole antibody conjugatesbecause they lack the Fc region, which would reduce nonspecificinteractions with Fc receptor-bearing cell membranes and would allowbetter penetration into tissues.

Labeled secondary antibodies of the invention may be used in signalamplification kits such as those commercialized by Molecular Probes,Inc. Such kits could each provide two labeled antibodies specific to aprimary antibodies, such as a mouse antibody. In one embodiment, arabbit anti-mouse IgG antibody conjugate of the invention is first usedto bind to the mouse-derived primary antibody. The fluorescence is thendramatically enhanced by the addition of a second conjugate of a goatanti-rabbit IgG antibody.

In yet another embodiment, the compounds of the invention may be used tolabel protein A and/or protein G. Protein A and protein G are bacterialproteins that bind with high affinity to the Fc portion of variousclasses and subclasses of immunoglobulins from a variety of species,such as Bovine, Cat, Chicken, Dog, Goat, Guinea pig, Horse, Human IgG1,IgG2, IgG3, IgG4, Human IgM, IgA, IgE, Human IgD, Mouse IgG1 or others,Pig, Rabbit, Rat or Sheep, which may be used in the detection ofimmunoglobulins. Alternatively, immunoglobins can be labeled with acompound of the invention having a structure of Formula I, II, III, IV,or V and retains binding specificity to its target after such labeling.These labeled immunoglobins can be used for in-vitro or in-vivodetection of the target antigen. In some embodiments, the labeledimmunoglobins comprise a fluorophore that has an absorption maximalwavelength equal to or greater than 750 nm. In other embodiments labeledimmunoglobins comprise a fluorophore that has an absorption maximalwavelength equal to or greater than 685 nm. In various embodiments ofthe invention, such labeled immunoglobins bind to an antigen on a cancercell. In some embodiments, the labeled immunoglobin binds to erb2.

Labeled antibodies prepared according to the invention may be primaryantibodies for various applications. While secondary detection methodscan provide significant signal amplification, a directly labeled primaryantibody often produces lower background fluorescence and lessnonspecific binding. Using primary antibodies also allows multipleprimary antibodies of the same isotype or derived from the same speciesto be used in the same experiment when they are directly labeled.

Examples of such primary antibodies include polyclonal antibodiesspecific for reporter gene products. These includeAnti-Green-Fluorescent Protein Antibodies, Anti-GlutathioneS-Transferase Antibody, Anti-beta-Glucuronidase Antibody,Anti-beta-Galactosidase Antibody, Monoclonal Antibodies Specific forEpitope Tags, Penta•His Antibody, Anti-HA Antibody and Anti-c-mycAntibody.

Organelle-specific labeled antibodies may also be prepared to labelvarious subcellular organelles and components such as the endoplasmicreticulum, peroxisomes, mitochondria, or cytochrome c. Labeledantibodies may also be specific for proteins in the oxidativephosphorylation system, such as antibodies against cytochrome oxidase(Complex IV) or antibodies against Complexes I, II, III and V, or othermitochondrial proteins such as anti-mitochondrial porin antibodies oranti-pyruvate dehydrogenase antibodies.

In other embodiments, labeled antibodies specific for proliferationmarkers and cell-cycle control proteins may be prepared. Such antibodiesinclude Anti-Bromodeoxyuridine Antibody (Anti-BrdU Antibody), which mayfor example be used in TUNEL assays, Anti-Human mRNA-Binding Protein HuRAntibody (Anti-HuR Antibody), Anti-Human Neuronal Protein HuC/HuDAntibody (Anti-Hu Antibody), Anti-cdc6 Peptide Antibody, Anti-CDAntibodies, Antibodies against D Cyclins/Cyclin-Dependent KinaseInhibitors, and Anti-Phosphoinositide Antibodies.

Some labeled antibodies may be specific for structural cellularproteins. Examples of such antibodies are Anti-alpha-Tubulin MonoclonalAntibody, Anti-Glial Fibrillary Acidic Protein (GFAP) Antibody,Anti-Desmin Antibody, or Anti-Fibronectin Antibody. Additionalantibodies suitable for use in the invention include antibodies specificfor neuronal proteins such as Anti-Synapsin I Antibody or Anti-NMDAReceptor Antibodies. Other Polyclonal and Monoclonal Antibodies that maybe labeled according to the invention include Anti-Human Golgin-97Antibody, Anti-Human Transferrin Receptor Antibody, Antibodies againstMatrix Metalloproteinases and Anti-Bovine Serum Albumin Antibody.

The specific interaction between an antigen and an antibody has beenexplored in the context of immunoassays utilizing the subjectfluorescent compounds. The immunoassays can permit single-moleculedetection or ensemble detection. The subject immunoassays can beperformed to characterize biological entities, screen for antibodytherapeutics, and determine the structural conformations of a targetantigen. For instance, immunoassays involving antibodies that arespecific for the biological entity or specific for a by-product producedby the biological entity have been routinely used to identify the entityby forming an antibody-entity complex. Immunoassays are also employed toscreen for antibodies capable of activating or down-regulating thebiological activity of a target antigen of therapeutic potential.Immunoassays are also useful for determining structural conformations byusing anti-idotypic antibodies capable of differentiating targetproteins folded in different conformations.

According to one embodiment of the invention, biomolecules labeled witha fluorescent group of the invention such as proteins are suitable forin vivo imaging, including without limitation imaging a biomoleculepresent inside a cell, a cell, tissue, organ or a whole subject. Wheredesired, the labeled biomolecules can be used to perform “In CellWestern” in which given molecules (e.g., a specific cellular protein)present inside a cell are stained and imaged.

The fluorescent groups of the invention and/or the labeled biomoleculesof the present invention can be administered to a subject in a varietyof forms adapted to the chosen route of administration, i.e., orally, orparenterally. Parenteral administration in this respect includes, but isnot limited to, administration by the following routes: intravenous,intramuscular, subcutaneous, parenteral, intraocular, intrasynovial,transepithelially including transdermal, opthalmic, sublingual, andbuccal; topically including opthalmic, dermal, ocular, rectal and nasalinhalation via insufflation and aerosol and rectal systemic. Inparticular, proteins labeled with a fluorescent group of the inventioncomprising an mPEG as a water soluble polymer group may be advantageous.In vivo imaging may provide means for early detection, screening,diagnosis, image-guided surgical intervention, and treatment of variousdiseases. For example, Near IR fluorescent group-labeled toxin (Veiseh,et al. Cancer Res. 67(14), 6882 (2007)) and antibody (Kulbersh, et al.Arch Otolaryngol Head Neck Surg. 133(5), 511 (2007) have been used todetect and guide the surgical removal of tumors. In in-vivo imaging, afluorescent probe, such as an antibody labeled with a fluorescent group,is first administered to an animal (such as a mammal). The animal isthen imaged by applying an excitation light with a wavelengthappropriate for the absorption of the fluorescent group and collectingthe fluorescence signal at another wavelength appropriate for theemission of the fluorescent group. Typically, for efficient tissuepenetration of both the excitation and emission lights, the absorptionand emission wavelengths of the fluorescent group may be greater than470 nm, greater than 550 nm, greater than 600 nm, or greater than 640nm. Absorption and emission wavelengths may be less than 1,200 nm.Fluorescent groups with wavelengths in the 640 nm-1,200 nm range may bereferred to as near infrared dyes, or near IR dyes, which are preferredfor tissue or in vivo imaging. An important challenge for in vivoimaging using antibodies has been the relatively short half-life of thefluorescently labeled antibodies. It has been reported that antibodieslabeled with more than 3 fluorescent group molecules were rapidlycleared from the body by translocating into the liver, where they becamemetabolized (BioProbes 52, 10-11, March 2007, by Molecular Probes, Inc).In order to extend the half-life of the labeled antibodies so thatenough of the antibodies were available over time for detecting thetarget, it was necessary to lower the number of fluorescent groupmolecules per antibody (i.e., degree of labeling or DOL) to about 2.However, the lowering of DOL was at the expense of fluorescencebrightness of the individual labeled antibody molecules. Thus, it wouldbe desirable to have antibodies that are labeled with 3 or morefluorescent group molecules and that have a relatively long half-life invivo. PEG is a known biocompatible material often used infunctionalizing the surface of implantable medical devices(Balakrishanan, et al. Biomaterials 26(17), 3495 (2005)) and inmodifying drugs (Mehvar, et al. Pharm. Pharmaceut. Sci. 3, 125 (2000);Wang, et al. J. Biochem. Cell Biology 34, 396 (2002)). In practice ofthe subject invention, proteins, such as antibodies, may be labeled withsingle or multiple, such as more than 3, 4, 5, 6 or more fluorescent dyemolecules of the invention and the antibodies labeled in such a mannercan have a relatively long half-life in the body. In particular, the PEGgroup(s) in the fluorescent group can mask the fluorescent group suchthat an antibody labeled with multiple molecules of the fluorescentgroup is less immunogenic as compare to the same antibody labeled with aconventional fluorescent dye (such as Cy5.5, Cy7 or Alexa Fluor 750). Insome aspects, PEG group(s) on the fluorescent group can mask or protectthe antibody itself, making the antibody more resistant to hydrolysis byproteases.

In other embodiments of the invention, a method of in-vivo imaging of asubject is provided comprising the steps of administering to a subjectin need thereof a biomolecule comprising a label having a structure ofFormula I, II, III, IV or V wherein the at least one reactive moiety oflabel has undergone a reaction which attached the label to thebiomolecule and wherein the biomolecule further comprises a targetingmoiety that binds to a binding partner on a cell of the subject which isindicative of the cell; binding the binding partner on the cell with thetargeting moiety of the biomolecule thereby differentially labeling thecell relative to neighboring cells; directing exciting wavelength to thecell; and detecting emitted fluorescence from the cell of the subjectthereby detecting the differentially labeled cell of the subject. Thebiomolecule may be an antibody, fragment of an antibody, protein,peptide, lipid or carbohydrate.

The compounds of the invention may also be used to produce labeledbiomolecules for use in immunohistochemistry and immunocytochemistryexperiments. In immunohistochemistry (IHC), the presence and location ofproteins is determined within a tissue section by exploiting theprinciple of an antibody binding specifically to an antigens present ina biological tissue. Such experiments may, for example, be used in thediagnosis and treatment of cancer. Specific molecular markers arecharacteristic of particular cancer types and are known to personsskilled in the art. IHC can also be used in basic research to determinethe distribution and localization of biomarkers in different parts of atissue. Visualization of antibody-antigen interactions can beaccomplished by reacting an antibody with a reactive fluorescentcompound of the invention and using the labeled antibody to stain tissuesections. In immunocytochemistry, the labeled antibody is used to stainpopulations of cultured cells. These techniques can be combined withconfocal laser scanning microscopy, which is highly sensitive and canalso be used to visualise interactions between multiple proteins.Subcellular localization of proteins may also be possible using confocalmicroscopy.

Of particular interest is the use of the labeled polypeptide forconducing immunocytochemistry. Fluorescence immunocytochemistry combinedwith fluorescence microscopy provides visualization of biomolecules suchas proteins and nucleic acids within a cell. One method uses primaryantibodies hybridized to the desired target. Then, secondary antibodiesconjugated with the subject fluorescent dyes and targeted to the primaryantibodies are used to tag the complex. The complex is visualized byexciting the dyes with a wavelength of light matched to the dye'sexcitation spectrum.

Immunocytochemistry can also be employed to discern subcellularlocalization of a given protein or nucleic acid. For instance,colocalization of biomolecules in a cell is performed using differentsets of antibodies for each cellular target. For example, one cellularcomponent can be targeted with a mouse monoclonal antibody and anothercomponent with a rabbit polyclonal antibody. These are designated as theprimary antibody. Subsequently, secondary antibodies to the mouseantibody or the rabbit antibody, conjugated to different fluorescentdyes of the present invention having different emission wavelengths, areused to visualize the cellular target.

The compounds of the invention or the labeled biomolecules of theinvention can also be used to label cells or particles for a variety ofapplications. Accordingly, the present invention provides a method ofindividually labeling a cell within a population of cells whereby thecell is differentially labeled relative to neighboring cells within thepopulation. The method typically comprises contacting the cell with alabeled biomolecule of the present invention, wherein said biomoleculecomprises a targeting moiety that binds to a binding partner that isindicative of said cell, and thereby differentially labeling the cellrelative to neighboring cells within the population. The targetingmoiety can be any biomolecules that recognize a binding partner on thecell to be detected. The choice of the targeting moiety will varydepending on the cell that is to be labeled. For example, for detectinga cancer cell, a targeting moiety is selected such that its bindingpartner is differentially expressed on a cancer cell. A vast number ofcancer markers are known in the art. They include without limitationcell surface receptors such as erb2, PDGF receptor, VEGF receptors, ahost of intracellular proteins such as phosphatidylinositol 3-kinases,c-abl, raf, ras, as well as a host of nuclear proteins includingtranscription factors and other nucleic acid binding molecules. In someother embodiments, the cancer marker is Immunoglobulin epsilon Fcreceptor II, Alk-1, CD20, EGF receptor, FGF receptor, NGF receptor,EpCam, CD3, CD4, CD11a, CD19, CD22, CD30, CD33, CD38, CD40, CD51, CD55,CD80, CD95, CCR2, CCR3, CCR4, CCR5, CTLA-4, Mucin 1, Mucin 16, Endoglin,Mesothelin receptor, Nogo receptor, folate receptor, CXCR4, insulin-likegrowth factor receptor, Ganglioside GD3, and alpha or beta Integrins. Todifferentially label various cell types, targeting moieties recognizinga cell-specific binding partner can be used. For example, there are ahost of protein markers differentially expressed on T cells as opposedon B cells or other cells of different lineage. Neuronal markers, musclecell markers, as well as markers indicative of cells of ectodermal,mesodermal or endodermal origins are also known in the art, all of whichcan be used depending on the intended applications. The targetingmoieties can be antibodies, receptors, cytokines, growth factors, andany other moieties or combinations thereof that are recognized by abinding partner on the cell to be labeled. The cell which is labeled maybe labeled intracellularly.

The differentially labeled cells can be imaged by directing excitingwavelength to the cell and detecting emitted fluorescence from the cell,in a number of in-vitro formats, either in solution or immobilized on asubstrate.

The labeled cells and/or the intensity of the fluorescence may bedetected or quantified by performing flow cytometry. Cells or particleslabeled with the compounds of the invention or stained with labeledbiomolecules of the invention may also be separated and isolated basedon the specific properties of the label using fluorescence activatedcell sorting (FACS). Such techniques are known in the art. Briefly,cells are labeled with a subject fluorescent dye and then passed, in asuspending medium, through a narrow dropping nozzle so that each cell istypically in a small droplet. A laser based detector system is used toexcite fluorescence and droplets with positively fluorescent cells aregiven an electric charge. Charged and uncharged droplets are separatedas they fall between charged plates and so collect in different tubes.The machine can be used either as an analytical tool, counting thenumber of labeled cells in a population or to separate the cells forsubsequent growth of the selected population. Further sophistication canbe built into the system by using a second laser system at right anglesto the first to look at a second fluorescent label or to gauge cell sizeon the basis of light scatter.

Additional guidance for performing fluorescent cell sorting can be foundin publications such as the following: Darzynkiewicz, Z., Crissman, H.A. and Robinson, J. P., Eds., Cytometry, Third Edition Parts A and B(Methods in Cell Biology, Volumes 63 and 64), Academic Press (2001);Davey, H. M. and Kell, D. B., “Flow cytometry and cell sorting ofheterogeneous microbial populations: the importance of single-cellanalyses,” Microbiological Rev 60, 641-696 (1996); Givan, A. L., FlowCytometry: First Principles, Second Edition, John Wiley and Sons (2001);Herzenberg, L. A., Parks, D., Sahaf, B., Perez, O., Roederer, M. andHerzenberg, L. A., “The history and future of the fluorescence activatedcell sorter and flow cytometry: a view from Stanford,” Clin Chem 48,1819-1827 (2002); Jaroszeski, M. J. and Heller, R., Eds., Flow CytometryProtocols (Methods in Molecular Biology, Volume 91), Humana Press(1997); Ormerod, M. G., Ed., Flow Cytometry: A Practical Approach, ThirdEdition, Oxford University Press (2000); Robinson, J. P., Ed., CurrentProtocols in Cytometry, John Wiley and Sons (1997); Shapiro, H. M.,“Optical measurement in cytometry: light scattering, extinction,absorption and fluorescence,” Meth Cell Biol 63, 107-129 (2001);Shapiro, H. M., Practical Flow Cytometry, Fourth Edition, Wiley-Liss(2003); Weaver, J. L., “Introduction to flow cytometry,” Methods 21,199-201 (2000).

Fluorescent compounds of the invention may also be used for fluorescencelifetime imaging (FLIM). FLIM is a useful technique for producing imagesbased on the variation in the fluorescence decay characteristics of afluorescent sample. It can be used as an imaging technique in confocalmicroscopy and other microscope systems. The lifetime of the fluorophoresignal, rather than its intensity, is used to create the image in FLIM,which has the advantage of minimizing the effect of photon scattering inthick layers of sample. FLIM may be useful for biomedical tissueimaging, allowing to probe greater tissue depths than conventionalfluorescence microscopy.

The compounds of the invention may be used in single moleculeapplications. Removal of ensemble averaging by observing individualmolecules of fluorescent group may allow the determination of themechanism of biological and chemical processes. Such processes mayinclude the translocation of protein motors such as kinesin or myosin,formation, dissolution and translocation of cellular protein complexesand the mechanism of action of DNA or RNA polymerases. In suchexperiments, the present compounds may be used, for example, to labelbiomolecules which are attached to a surface such as a microscopy slideor flow chamber. Individual fluorophores may subsequently be observedusing total internal reflection fluorescence microscopy.

The present compounds may also be used for the labeling of lipids.Lipids are involved in many biological processes, and the labeling oflipids and lipid rafts may is often a valuable method for studying theirproperties. Various lipid monolayers and bilayers may be labeled in livecells or artificial systems such as liposomes and micelles. For example,a live cell population may be labeled with a fluorescent conjugateprepared by reacting a compound of the invention and cholera toxinsubunit B, which specifically interacts with lipid rafts. Such lipidrafts may then be crosslinked into distinct membrane patches by the useof an anti-cholera toxin antibody, which may be labeled with one of thepresent compounds.

The labeled polypeptides of the present invention find use as biosensorsin prokaryotic and eukaryotic cells, e.g. as calcium ion indicators, aspH indicators, as phorphorylation indicators, as indicators of otherions including without limiting to magnesium, sodium, potassium,chloride and halides. For example, for detection of calcium ion,proteins containing an EF-hand motif are known to translocate from thecytosol to membranes upon binding to calcium ion. These proteins containa myristoyl group that is buried within the molecule by hydrophobicinteractions with other regions of the protein. Binding of calcium ioninduces a conformational change exposing the myristoyl group which thenis available for the insertion into the lipid bilayer. Labeling such anEF-hand containing protein with a subject fluorescent dye makes it anindicator of intracellular calcium ion concentration by monitoring thetranslocation from the cytosol to the plasma membrane. Such monitoringcan be performed with the use of an optical detector, e.g., a confocalmicroscope. EF-hand proteins suitable for use in this system include,but are not limited to: recoverin (1-3), calcineurin B, troponin C,visinin, neurocalcin, calmodulin, parvalbumin, and the like.

For use as a pH indicator, a system based on hisactophilins may beemployed. Hisactophilins are myristoylated histidine-rich proteins knownto exist in Dictyostelium. Their binding to actin and acidic lipids issharply pH-dependent within the range of cytoplasmic pH variations. Inliving cells membrane binding seems to override the interaction ofhisactophilins with actin filaments. At pH of approximately 6.5 theytypically locate to the plasma membrane and nucleus. In contrast, at pH7.5 they evenly distribute throughout the cytoplasmic space. This changeof distribution is reversible and is attributed to histidine clustersexposed in loops on the surface of the molecule. The reversion ofintracellular distribution in the range of cytoplasmic pH variations isin accord with a pK of 6.5 of histidine residues. The cellulardistribution is independent of myristoylation of the protein. Byconjugating the subject fluorescent dye to hisactophilin, theintracellular distribution of the labeled hisactophilin can be followedby laser scanning, confocal microscopy or standard fluorescencemicroscopy. Quantitative fluorescence analysis can be done by performingline scans through cells (laser scanning confocal microscopy) or otherelectronic data analysis (e.g., using metamorph software (UniversalImaging Corp) and averaging of data collected in a population of cells.

The subject fluorescent proteins also find use in applications involvingthe automated screening of arrays of cells by using microscopic imagingand electronic analysis. Screening can be used for drug discovery and inthe field of functional genomics: e.g., where the subject proteins areused as markers of whole cells to detect changes in multicellularreorganization and migration, e.g., formation of multicellular tubules(blood vessel formation) by endothelial cells, migration of cellsthrough Fluoroblok Insert System (Becton Dickinson Co.), wound healing,neurite outgrowth; where the proteins are used as markers fused topeptides (e.g., targeting sequences) and proteins that allow thedetection of change of intracellular location as indicator for cellularactivity, for example: signal transduction, such as kinase andtranscription factor translocation upon stimuli, such as protein kinaseC, protein kinase A, transcription factor NFkB, and NFAT; cell cycleproteins, such as cyclin A, cyclin B1 and cyclinE; protease cleavagewith subsequent movement of cleaved substrate, phospholipids, withmarkers for intracellular structures such as endoplasmic reticulum,Golgi apparatus, mitochondria, peroxisomes, nucleus, nucleoli, plasmamembrane, histones, endosomes, lysosomes, microtubules, actin.

The subject fluorescent proteins also find use in high through-putscreening assays. The subject fluorescent proteins are typically morestable than proteins lacking the subject fluorescent dyes. In someaspects, the fluorescent proteins can exhibit a serum half-life of morethan 1 hour, 2 hours, 5 hours, or 24 hours or more.

The subject fluorescent proteins can be used as second messengerdetectors, e.g., by conjugating the subject fluorescent dyes to specificsignaling domains, e.g., calcium binding SH2-, SH3-, PH-, PDZ-domain andetc.

The examples below are for the purpose of illustrating the practice ofthe invention. They shall not be construed as being a limitation on thescope of the invention or claims.

EXAMPLES Example 1 Preparation of2-(anilinovinyl)-1-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindolium,Potassium Salt (Compound No. 1)

A mixture of1-(5-carboxypentyl)-2,3,3-trimethylindoleninium-5-sulfonate, potassiumsalt (Bioconjugate Chem. 4, 105 (1993)) (5 g), N,N′-diphenylformamidine(2.3 g) and acetic anhydride (1.1 mL) was heated at 120° C. for 30minutes. After cooling down to room temperature, the mixture wasconcentrated to dryness under vacuum and the residue was purified bycolumn chromatography on silica gel (3.5 g).

Example 2 Preparation of2-(4-anilinobutadienyl)-1-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindolium,Potassium Salt (Compound No. 2)

A mixture of1-(5-carboxypentyl)-2,3,3-trimethylindoleninium-5-sulfonate, potassiumsalt (10 g), malonaldehyde dianil hydrochloride (8 g), Et₃N (0.356 mL)in AcOH (40 mL) was heated at 120° C. for 3 hours. After cooling down toroom temperature, the mixture was concentrated to dryness under vacuumand the residue was purified by column chromatography on silica gel todark brown gummy solid (5 g).

Example 3 Preparation of Compound Nos. 3a and 3b

To a solution of1-(5-carboxypentyl)-2,3,3-trimethylindoleninium-5-sulfonate, potassiumsalt (0.1 g) in DMF (1 mL) was added Et₃N (0.1 mL) and TSTU (80 mg). Themixture was stirred at room temperature for 1 hour, followed by theaddition of Et₃N (40 μL) and m-dPEG₂₄ amine (0.4 g) (QuantaBiodesign,Powell, Ohio) or m-dPEG₁₂ amine (0.2 g) (QuantaBiodesign, Powell, Ohio).The mixture was stirred at room temperature overnight and thenconcentrated to dryness. The residue was purified by columnchromatography on silica gel to give a light brown solid (250 mg forcompound No. 3a and 102 mg for compound No. 3b).

Example 4 Preparation of Compound No. 4

A mixture of compound No. 3a (40 mg), compound No. 1 (30 mg), aceticanhydride (15 μL) and Et₃N (45 μL) in DMF (1 mL) was stirred at roomtemperature overnight. The dark red solution was concentrated to drynessunder vacuum and the residue was purified by column chromatography onsilica gel to give a dark red solid (40 mg).

Example 5 Preparation of Compound No. 5

A mixture of compound No. 4 (15 mg), Et₃N (3.5 μL) and TSTU (3 mg) inDMF (0.2 mL) was stirred at room temperature for 1 hour. Et₂O (5 mL) wasadded and the precipitate (19 mg) was collected by centrifugation.

Example 6 Preparation of Compound No. 6

A mixture of compound No. 3a (30 mg), compound No. 2 (20 mg), aceticanhydride (10 μL) and Et₃N (30 μL) in DMF (1 mL) was stirred at roomtemperature overnight. The dark blue solution was concentrated todryness under vacuum and the residue was purified by columnchromatography on silica gel to give a dark blue solid (20 mg).

Example 7 Preparation of Compound No. 7

To a solution of compound No. 6 (13 mg) in DMF (0.2 mL) was added Et₃N(3 μL) and TSTU (2.5 mg) and the mixture was stirred at room temperaturefor 1 hour. Et₂O (2 mL) was added and the precipitate (23 mg) wascollected by centrifugation.

Example 8 Preparation of Compound No. 8

Compound 8 (60 mg) was synthesized from compound No. 2 (35 mg) andcompound No. 3b (50 mg) according to the preparation of compound No. 6.

Example 9 Preparation of Compound No. 9

Compound No. 9 (10 mg) was synthesized from compound No. 8 (14 mg)according to the preparation of compound No. 7.

Example 10 Preparation of Compound No. 10

Compound No. 10 (75 mg) was prepared from1-(5-carboxypentyl)-2,3,3-trimethylindoleninium-5-sulfonate, potassiumand mPEG-NH₂ (Mwt˜2,000) (Laysan Bio. Arab, Ala.) according to thesynthesis of compound No. 3.

Example 11 Preparation of Compound No. 11

Compound No. 11 (15 mg) was prepared from compound No. 10 (20 mg) andcompound No. 2 (6 mg) according to the synthesis of compound No. 6.

Example 12 Preparation of Compound No. 12

Compound No. 12 (3 mg) was prepared from compound No. 11 (4 mg)according to the synthesis of compound No. 7.

Example 13 Preparation of Compound No. 13

A mixture of p-hydrazinobenzenesulfonic acid (5 g), methyl7-methyl-8-oxononanoate (6 g) (US patent application 2006/0121503 A1) inacetic acid (20 mL) was refluxed gently for 3 hours. After cooling downto room temperature, the mixture was concentrated to dryness and theresidue was purified by column chromatography on silica gel to give areddish brown solid (3 g). The solid was mixed with NaOAc (1 equivalent)in MeOH (100 mL) and the resulting solution was stirred at roomtemperature for 30 minutes. The solution was concentrated to drynessunder vacuum to give a reddish brown solid (3 g).

Example 14 Preparation of Compound No. 14

A mixture of compound No. 13 (0.86 g) and 1,3-propanesulftone (0.83 g)was heated at 120° C. for 3 hours. EtOAc (50 mL) was added and thesuspension was refluxed gently for 2 hours. After cooling to roomtemperature, the precipitate (1 g) was collected by suction filtration.

Example 15 Preparation of Compound No. 15

A mixture of compound No. 14 (0.8 g), malonaldehyde dianil hydrochloride(0.52 g), Et₃N (23 μL) in AcOH (3 mL) was heated at 120° C. for 3 hours.After cooling down to room temperature, the mixture was concentrated todryness under vacuum and the residue was purified by columnchromatography on silica gel to dark red gummy solid (0.5 g).

Example 16 Preparation of Compound No. 16

Compound No. 16 (80 mg) was prepared from compound No. 15 (50 mg) andcompound No. 3a (120 mg) according to the synthesis of compound No. 6.

Example 17 Preparation of Compound No. 17

To a solution of compound No. 16 (50 mg) in H₂O (2 mL) is added 1 M NaOH(120 μL). The solution was stirred at room temperature for 1 hour andthen purified by LH-20 column (35 mg).

Example 18 Preparation of Compound No. 18

Compound No. 18 (5 mg) was prepared from compound No. 17 (4 mg)according to the synthesis of compound No. 7.

Example 19 Preparation of Compound No. 19

A mixture of compound No. 13 (0.6 g) and 6-bromohexanoic acid (0.63 g)was heated at 140° C. for 1 hour. EtOAc (30 mL) was added and thesuspension is refluxed gently for 1 hour and the precipitate (0.45 g)was collected by suction filtration.

Example 20 Preparation of Compound No. 20

A mixture of compound No. 19 (84 mg), compound No. 2 (52 mg), aceticanhydride (32 μL) and Et₃N (0.1 mL) in DMF (2 mL) was stirred at roomtemperature overnight. The solution was concentrated to dryness undervacuum and the residue was purified by column chromatography on silicagel to give dark blue solid (20 mg).

Example 21 Preparation of Compound No. 21

Compound No. 21 (17 mg) was prepared from compound No. 20 (9 mg) andm-dPEG₂₄ amine (27 mg) according to the synthesis of compound No. 3.

Example 22 Preparation of Compound No. 22

To a solution of compound No. 21 (17 mg) in H₂O (0.5 mL) was added 1 MNaOH (0.1 mL) and the solution was stirred at room temperature for 30minutes. The solution was acidified with 1 N HCl (0.1 mL) and purifiedby LH-20 column to give a dark blue solid (10 mg) after lyophilization.

Example 23 Preparation of Compound No. 23

Compound No. 23 (4 mg) was prepared from compound No. 22 (6 mg)according to the synthesis of compound No. 7.

Example 24 Preparation of Compound Nos. 24a and 24b

To a solution of undecaethylene glycol methyl ether (1 g) (Polypure AS,Oslo, Norway) or m-dPEG₂₄ alcohol (1 g) (QuantaBiodesign, Powell, Ohio)in CH₂Cl₂ (5 mL) and pyridine (5 mL) at 0° C. is added p-TsCl (1.1equivalents) portionwise. The mixture was stirred at 0° C. for 2 hoursand then at room temperature overnight. The solution was concentrated todryness in vacuo and the residue is purified by column chromatography onsilica gel to give a colorless oil (1.25 g for compound No. 24a and 1.10g for compound No. 24b).

Example 25 Preparation of Compound No. 25a and 25b

H₃CO—R—OCH₂CH₂I

Compound No. 25a: R=—(CH₂CH₂O)₁₀CH₂CH₂—

Compound No. 25b: R=—(CH₂CH₂O)₂₃CH₂CH₂—

A mixture of compound No. 24a (1.2 g) or compound No. 24b (1.1 g) andNaI (1.1 equivalent) in acetone (10 mL) was refluxed gently overnight.After cooling down to room temperature the mixture was concentrated todryness under vacuum and the residue was purified by columnchromatography on silica gel to give a colorless solid (1.1 g forcompound No. 25a and 1 g for compound No. 25b).

Example 26 Preparation of Compound No. 26

A mixture of sodium ethoxide (0.13 g) and ethyl 2-methylacetoacetate(0.28 g) in anhydrous EtOH (5 mL) was stirred at room temperature for 1hour, followed by the addition of compound No. 25a (0.8 g). The mixturewas refluxed gently overnight and the solution was concentrated todryness under vacuum. The residue was purified by column chromatographyon silica gel to give an off-white oil (0.75 g).

Example 27 Preparation of Compound No. 27

To a solution of compound No. 26 (0.7 g) in MeOH (10 mL) was added asolution of NaOH (0.21 g) in H₂O (2 mL). The mixture was heated at 60°C. overnight. After cooling down to room temperature, the solution wasneutralized with 6M HCl (1 mL). The solution was concentrated to drynessunder vacuum and the residue was purified by column chromatography onsilica gel to give a colorless oil (0.55 g).

Example 28 Preparation of Compound No. 28

A mixture of p-hydrazinobenzenesulfonic acid (100 mg) and compound No.27 (300 mg) in acetic acid (5 mL) was heated to reflux overnight. Aftercooling down to room temperature, the mixture was concentrated todryness and the residue was purified by column chromatography on silicagel a pale brown solid (270 mg). The solid was mixed with NaOAc (1equivalent) in MeOH (10 mL) and the resulting solution was stirred atroom temperature for 30 minutes. The solution was concentrated todryness under vacuum to give reddish brown solid (280 mg).

Example 29 Preparation of Compound No. 29

A mixture of compound No. 28 (200 mg) and large excess of ethyl iodide(10 mL) was heated to boiling overnight. EtOAc (20 mL) was added and thesuspension was refluxed gently for 1 hour. After cooling to roomtemperature, the precipitate (250 mg) was collected by suctionfiltration.

Example 30 Preparation of Compound No. 30

Compound No. 30 (55 mg) was prepared from compound No. 29 (100 mg) andcompound No. 2 (65 mg) according to the synthesis of compound No. 6.

Example 31 Preparation of Compound No. 31

Compound No. 31 (120 mg) was prepared from compound No. 30 (200 mg) and6-bromohexanoic acid (1 g) according to the synthesis of compound No.19.

Example 32 Preparation of Compound No. 32

Compound No. 32 (75 mg) was prepared from compound No. 31 (100 mg)according to the synthesis of compound No. 2.

Example 33 Preparation of Compound No. 33

Compound No. 33 (7 mg) was prepared from compound No. 32 (30 mg) andcompound No. 3a (45 mg). according to the synthesis of compound No. 6.

Example 34 Preparation of Compound No. 34

Compound No. 34 (3 mg) was prepared from compound No. 33 (5 mg)according to the synthesis of compound No. 7.

Example 35 Preparation of Compound No. 35

Compound No. 35 (7 mg) was prepared from compound No. 32 (30 mg) andcompound No. 29 (23 mg) according to the synthesis of compound No. 6.

Example 36 Preparation of Compound Nos. 36a and 36b

Compound No. 36a (110 mg) and compound No. 36b (100 mg) were eachprepared by quaternizing 2,3,3-trimethylindoleninium-5-sulfonate, sodiumsalt (1 equivalent) with compound No. 25a (600 mg) and compound No. 25b(400 mg), respectively, according to the synthesis of compound No. 19.

Example 37 Preparation of Compound No. 37

Compound No. 37 (11 mg) was prepared from compound No. 36a (25 mg) andcompound No. 32 (40 mg) according to the synthesis of compound No. 6.

Example 38 Preparation of Compound No. 38

Compound No. 38 (36 mg) was synthesized from compound No. 13 (15 mg) andcompound No. 25b (50 mg) according to the preparation of compound No.19.

Example 39 Preparation of Compound No. 39

Compound No 39 (70 mg) was synthesized from compound No. 36b (100 mg)according to the preparation of compound No. 2.

Example 40 Preparation of Compound No. 40

Compound No. 40 (38 mg) was synthesized from compound No. 38 (30 mg) andcompound No. 39 (32 mg) according to the preparation of compound No. 6.

Example 41 Preparation of Compound No. 41

Compound No. 40 (20 mg) was hydrolyzed to give the free acid form (14mg) according to the synthesis of compound No. 22. The free acid form ofthe dye was then converted to compound No. 41 (10 mg) according to thepreparation of compound No. 23.

Example 42 Preparation of Compound No. 42

Compound No. 42 (0.19 mg) was synthesized from1-(5-carboxypentyl)-2,3,3-trimethylindoleninium bromide (0.1 g) andm-dPEG₂₄ amine (0.3 g) according to the preparation of compound No. 3b.

Example 43 Preparation of Compound No. 43

Compound No. 43 (0.5 g) was synthesized from1-(5-carboxypenthyl)-2,3,3-trimethylindoleninium bromide (1 g) andmalonaldehyde dianil hydrochloride (0.85 g) according to the preparationof compound No. 2.

Example 44 Preparation of Compound No. 44

Compound No. 44 (25 mg) was prepared from compound No. 42 (40 mg) andcompound No. 43 (15 mg) according to the synthesis of compound No. 6.

Example 45 Preparation of Compound No. 45

Compound No. 45 (20 mg) was prepared from compound No. 44 (25 mg)according to the synthesis of compound No. 7.

Example 46 Preparation of Compound No. 46

To sodium hydride (300 mg) in DMF at 0° C. was added5,6-dichloro-2-methylbenzoimidazole (500 mg) in one portion. The mixturewas stirred at 0° C. for 15 minutes, followed by addition of ethyl6-bromohexanoate (0.66 mL). The mixture was stirred at 0° C. for another15 minutes and then at room temperature for 1 hour. The solution wasconcentrated to dryness under vacuum and the residue was purified bycolumn chromatography on silica gel to give pale brown solid (0.75 g).

Example 47 Preparation of Compound No. 47

A mixture of compound No. 46 (170 mg) and 6-bromohexanoic acid (200 mg)was heated at 140° C. for 1 hour. EtOAc (20 mL) was added and thesuspension was refluxed gently for 30 minutes. After cooling down toroom temperature, the precipitate (260 mg) was collected by suctionfiltration.

Example 48 Preparation of Compound No. 48

Compound No. 48 (55 mg) was synthesized from compound No. 47 (0.1 g) andm-dPEG24 amine (200 mg) according to the preparation of compound No. 3a.

Example 49 Preparation of Compound No. 49

Compound No. 49 (700 mg) was prepared from2-methyl-6-t-butylbenzooxazole (540 mg) and 1,3-propanesulftone (450 mg)according to the synthesis of compound No. 14.

Example 50 Preparation of Compound No. 50

Compound No. 50 (270 mg) was prepared from compound No. 49 (460 mg) andN,N′-diphenylformamidine (340 mg) according to the synthesis of compoundNo. 1.

Example 51 Preparation of Compound No. 51a

Compound No. 48 (30 mg) and compound No. 50 (10 mg) were coupled to givea cyanine dye ethyl ester intermediate (16 mg) according to thesynthesis of compound No. 4. The resulting intermediate was hydrolyzedusing 1 M NaOH to give the free acid dye compound No. 51 (10 mg)according to the synthesis of compound No. 22.

Example 52 Preparation of Compound No. 52

Compound No. 52 (7 mg) was prepared from compound No. 51 (8 mg)according to the synthesis of compound No. 5.

Example 53 Preparation of Compound No. 53

Compound No. 53 (18 g) was prepared from2,3,3-trimethylindoleninium-5-sulfonate, sodium salt (11 g)(Bioconjugate Chem. 4, 105 (1993)) according to the synthesis ofcompound No. 49.

Example 54 Preparation of Compound No. 54

Compound No. 54 (2.4 g) was prepared from compound No. 53 (5.7 g)according to the synthesis of compound No. 1.

Example 55 Preparation of Compound No. 55

Compound No. 54 (19 mg) and compound No. 48 (33 mg) were coupled to givea cyanine dye ethyl ester intermediate (25 mg) according to thesynthesis of compound No. 4. Hydrolysis of the intermediate using 1 MNaOH give compound No. 55 (17 mg) according to the synthesis of compoundNo. 22.

Example 56 Preparation of Compound No. 56

Compound No. 56 (6 mg) was prepared from compound No. 55 (10 mg)according to the synthesis of compound No. 5.

Example 57 Preparation of Compound No. 57

Compound No. 57 (55 mg) was synthesized from compound No. 19 (80 mg) andm-dPEG24 amine (100 mg) according to the preparation of compound No. 3a

Example 58 Preparation of Compound No. 58

Compound No. 58 (8 mg) was prepared from compound No. 57 (20 mg) andcompound No. 50 (10 mg) according to the synthesis of compound No. 4.

Example 59 Preparation of2-(6-anilinohexatrienyl)-1-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindolinium,Inner Salt (Compound No. 59)

Compound No. 59 (4 g) was prepared from1-(5-carboxypentyl)-2,3,3-trimethylindoleninium-5-sulfonate inner salt(12 g) and glutoconaldehyde dianil hydrochloride (18 g) according to thesynthesis of compound No. 2.

Example 60 Preparation of Compound No. 60

Compound No. 60 (15 mg) was prepared from compound No. 59 (50 mg) andcompound No. 3a (50 mg) according to the synthesis of compound No. 6.

Example 61 Preparation of Compound No. 61

Compound No. 61 (6 mg) was prepared from compound No. 60 (10 mg)according to the synthesis of compound No. 7.

Example 62 Preparation of Compound No. 62

Compound No. 62 (5.37 g) was prepared from 6-hydrazinonaphthalene1,3-disulfonate (5.3 g) (Bioconjugate Chem. 7, 356 (1996)) and methyl7-methyl-8-oxononanoate (3.8 g) according to the synthesis of compoundNo. 13.

Example 63 Preparation of Compound No. 63

Compound No. 63 (15 g) was prepared from compound No. 62 (5.37 g),1,3-propanesulftone (6 g) and according to the synthesis of compound No.14.

Example 64 Preparation of Compound No. 64

Compound No. 64 (1.5 g) was prepared from compound No. 63 (7 g)according to the synthesis of compound No. 2.

Example 65 Preparation of Compound No. 65

Compound No. 65 (17 mg) was prepared from compound No. 64 (60 mg) andcompound No. 3a (100 mg) according to the synthesis of compound No. 6.

Example 66 Preparation of Compound No. 66

Compound No. 66 (6 mg) was prepared from compound No. 72 (15 mg)according to the synthesis of compound No. 17.

Example 67 Preparation Compound No. 67

Compound No. 67 (3 mg) was prepared from compound No. 66 (5 mg)according to the synthesis of compound No. 7.

Example 68 Preparation of Compound No. 68

A mixture of compound No. 31 (86 mg), compound No. 36a (76 mg) andsquaric acid (12 mg) in 1-butanol:toluene (10 mL, 1:1) was heated torefluxed overnight using a Dean-Stark trap filled with 4 Å molecularsieve. After cooling down to room temperature, the solution wasconcentrated to dryness under vacuum and the residue was purified bycolumn chromatography on silica gel to give a dark blue solid (8 mg).

Example 69 Preparation of Compound No. 69

Compound No. 69 (60 mg) was prepared from compound No. 31 (100 mg)according to the synthesis of compound No. 1.

Example 70 Preparation of Compound No. 70

Compound No. 70 (50 mg) was prepared from compound No. 36a (100 mg)according to the synthesis of compound No. 1.

Example 71 Preparation of Compound No. 71

A mixture of compound No. 69 (40 mg), compound No. 70 (36 mg),3-pyrrolidino-1-cyclopentene (6 mg), acetic anhydride (12 μL) and Et₃N(30 μL) in DMF (1 mL) was stirred at room temperature overnight. Thesolution is concentrated to dryness under vacuum and the residue waspurified by column chromatography on silica gel to give greenish solid(10 mg).

Example 72 Preparation of Compound No. 72

A mixture of compound No. 31 (45 mg), compound No. 36a (40 mg),2-chloro-3-(anilinomethylene)-1-(aniliniummethyl)cyclohex-1-ene (17 mg),acetic anhydride (16 μL) and Et₃N (38 μL) in DMF (1 mL) was stirred atroom temperature overnight. The solution was concentrated to drynessunder vacuum and the residue was purified by column chromatography onsilica gel to give greenish solid (9 mg).

Example 73 Preparation of Compound No. 73

To a solution of Boc-Glu(OBut)-OH (30 mg) (Advanced ChemTech,Louisville, Ky.) in DMF (1 mL) was added Et₃N (42 μL) and TSTU (30 mg).The mixture was stirred at room temperature for 3 hours, followed by theaddition of m-dPEG₂₄ amine (100 mg). The mixture was kept stirring atroom temperature overnight and then concentrated to dryness undervacuum. The residue was purified by column chromatography on silica gelto give compound No. 73 as a colorless oil (70 mg).

Example 74 Preparation of Compound No. 74

To a solution of compound No. 73 (68 mg) in CH₂Cl₂ (1 mL) at 0° C. wasadded TFA (1 mL). The mixture was stirred at 0° C. for 1 hour and thenat room temperature overnight. The solution was concentrated to drynessunder vacuum to give a colorless oil (68 mg).

Example 75 Preparation of Compound No. 75

To a suspension of 5-(and -6)-carboxyrhodamine 110, succinimidyl ester(14 mg) (Biotium, Hayward, Calif.) in DMF (500 μL) at room temperaturewas added Et₃N (50 μL) and compound No. 74 (34 mg). The mixture wasstirred at room temperature overnight and then concentrated to drynessunder vacuum. The residue was purified by LH-20 column (24 mg).

Example 76 Preparation of Compound No. 76

To a solution of compound No. 75 (24 mg) in DMF (500 μL) at 0° C. wasadded Et₃N (10 μL) and TSTU (4.5 mg). The mixture was stirred at 0° C.for 1 hour, followed by the addition of Et₃N (5 μL) and a solution of6-amino-1-hexanoic acid (4 mg) in H₂O (100 μL). The mixture was stirredat room temperature overnight and then concentrated to dryness undervacuum. The residue was purified by LH-20 column to give a yellow solid(10 mg).

Example 77 Preparation of Compound No. 77

To a solution of compound No. 76 (10 mg) in DMF (500 μL) at 0° C. wasadded Et₃N (3 μL) and TSTU (2 mg). The mixture was stirred at 0° C. for1 hour and Et₂O (3 mL) was added. The precipitate (5 mg) was collectedby centrifugation.

Example 78 Preparation of Compound No. 78

To a solution of 7-amino-4-methylcoumarin-3-acetic acid, succinimidylester (20 mg, prepared according to U.S. Pat. No. 4,956,480) in DMF (500μL) was added Et₃N (50 μL) and compound No. 74 (34 mg). The mixture wasstirred at room temperature for 2 hours and then concentrated to drynessunder vacuum. The residue was purified by LH-20 column (30 mg).

Example 79 Preparation of Compound No. 79

Compound 79 (10 mg) was prepared from compound No. 78 (15 mg) accordingto the synthesis of compound No. 76.

Example 80 Preparation of Compound No. 80

Compound No. 80 (7 mg) was prepared compound No. 79 (10 mg) according tothe synthesis of compound No. 77.

Example 81 Preparation of Compound No. 81

Compound No. 81 (12 mg) was prepared from1,3,6-trisulfo-8-pyrenyloxyacetyl azide, sodium salt (15 mg, U.S. Pat.No. 5,132,432) and compound No. 74 (15 mg) according to the synthesis ofcompound No. 75.

Example 82 Preparation of Compound 82

Compound No. 82 (6 mg) was prepared from compound No. 81 (14 mg)according to the synthesis of compound No. 76.

Example 83 Preparation of Compound No. 83

Compound No. 83 (5 mg) was prepared from compound No. 82 (5 mg)according to the synthesis of compound No. 77.

Example 84 Preparation of Compound No. 84

A mixture of compound No. 72 (8 mg) and sodium phenoxide (10 mg) washeated at 70° C. overnight and then concentrated to dryness undervacuum. The residue was purified by LH-20 column (3 mg).

Example 85 Preparation of Compound No. 85

To a solution of compound No. 7 (5 mg) in DMF (200 μL) was added Et₃N (5μL) and N-(5-aminopentyl)maleimide, trifluoroacetate salt (4 mg,Biotium). The mixture was stirred at room temperature for 1 hour andthen concentrated to dryness under vacuum. The residue was purified bycolumn chromatography on silica gel (2 mg).

Example 86 Preparation of Compound No. 86

To a solution of compound No. 7 (5 mg) in DMF (200 μL) was addedanhydrous hydrazine (10 μL). The mixture was stirred at room temperaturefor 2 hours and then acidified with 1N HCl. The solution wasconcentrated to dryness under vacuum and the residue was purified byLH-20 column (3 mg).

Example 87 Preparation of Compound No. 87

Compound No. 87 (5 mg) was prepared from compound No. 7 (10 mg) and monot-BOC-cadaverine (2 equivalents) according to the synthesis of compoundNo. 85.

Example 88 Preparation of Compound No. 88

To a solution of compound No. 87 (4 mg) in CH₂Cl₂ (500 μL) at 5° C. wasadded TFA (250 μL). The mixture was stirred at 5° C. for 30 minutes andthen concentrated to dryness under vacuum. The reside was purified byLH-20 column to give a dark blue solid (1.5 mg)

Example 89 Preparation of Compound No. 89

A mixture of compound No. 24a (1 g), methyl 6-aminohexanoate (0.32 g)and diisopropylethylamine (0.61 mL) in CH₃CN (5 mL) was refluxed gentlyovernight. After cooling down to room temperature, the solution wasconcentrated to dryness under vacuum. The residue was purified by silicagel column to give a pale yellow oil (0.4 g).

Example 90 Preparation of Compound No. 90

To a solution of 7-amino-4-methylcoumarin-3-acetic acid, succinimidylester (17 mg) was added Et₃N (50 μL) and compound No. 89 (60 mg) in DMF(0.5 mL). The mixture was stirred at room for 30 minutes and thenconcentrated to dryness under vacuum. The residue was purified by silicagel column (46 mg).

Example 91 Preparation of Compound No. 91

To a solution of compound No. 90 (40 mg) in H₂O (0.5 mL) was added 1MNaOH solution (0.15 mL). The mixture was stirred at room temperature for1 hr and then acidified with 1 M HCl (0.15 mL). The aqueous solution waspurified by LH-20 column. (35 mg).

Example 92 Preparation of Compound No. 92

Compound No. 92 (30 mg) was prepared from compound No. 91 (34 mg)according to the synthesis of compound No. 80.

Example 93 Preparation of Compound No. 93

Compound No. 93 (40 mg) was prepared from7-amino-4-methyl-6-sulfocoumarin-3-acetic acid succinimidyl ester (16mg) (Bioorg. & Med. Chem. Letters, 9, 2229 (1999)) according to thesynthesis of compound No. 90.

Example 94 Preparation of Compound No. 94

Compound No. 94 (20 mg) was prepared from compound No. 93 (35 mg)according to the synthesis of compound No. 91.

Example 95 Preparation of Compound No. 95

Compound No. 95 (15 mg) was prepared from compound No. 94 (17 mg)according to the synthesis of compound No. 80.

Example 96 Preparation of Compound No. 96

To a mixture of 5-(and -6)-carboxyrhodamine 110, succinimidyl ester(CR110 SE) (60 mg) (Biotium, Hayward, Calif.) and Et₃N (90 μL) in DMF(500 μL) was added compound No. 89 (68 mg). The mixture was stirred atroom temperature for 2 days and then concentrated to dryness undervacuum. The residue was redissolved in H₂O (500 μL) and 1N NaOH (400 μL)was added. The mixture was stirred at room temperature overnight andthen loaded onto a LH 20 column. Eluting of the LH-20 column with waterproduced pure free acid form of the rhodamine dye (45 mg). The free acidrhodamine dye (30 mg) was converted to compound No. 96 (22 mg) accordingto the synthesis of compound No. 80.

Example 97 Preparation of Protein Dye-Conjugates

Fluorescent conjugates of goat anti-mouse IgG (GAM), goat anti-rabbitIgG (GAR), and streptavidin were prepared from the respective proteinsand a reactive dye, following published procedures (U.S. Pat. No.6,974,873; Haugland et al., Meth. Mol. Biol. 45, 205 (1995); Haugland etal., Meth. Mol. Biol. 45, 223 (1995); Haugland et al., Meth. Mol. Biol.45, 235 (1995); Haugland et al., Current Protocols in Cell Biology,16.5.1-16.5.22 (2000)). Briefly, an antibody or streptavidin at 1 mg/mLin 0.1 mM pH 8.5 sodium bicarbonate buffer was mixed with one of thereactive dye at various ratio of dye molecules/protein molecule. Afterincubating for about an hour at room temperature, the reaction mixturewas separated by gel filtration using Sephadex G-25 equilibrated withPBS (pH 7.4). The various dye molecules/protein ratios used in thelabeling reactions produced protein conjugates with different degree ofdye labeling (DOL) as listed in Table 7 below for each dye/protein pair.

TABLE 7 List of selected antibody and streptavidin conjugates preparedaccording to the invention Degree of Protein Dye Labeling (DOL)Streptavidin Compound 2.8; 3.8; 4.6; 7.8; 9.1; 9.4; 10.6 No. 5 Goatanti-mouse IgG Compound 1.1; 1.7; 2.9; 3.7; 4.9; 5.8; 7.4; 7.6 No. 5Goat anti-rabbit IgG Compound 1.4; 1.9; 3.0; 5.2; 6.1; 7.1; 9.3 No. 41Goat anti-mouse IgG Compound 1.1; 1.7; 2.7; 3.9; 4.8; 5.8; 6.6; 7.3 No.41 Goat anti-mouse IgG Compound 1.7; 3.5; 4.9; 6.2; 8.3 No. 67 Goatanti-mouse IgG Compound 1.7; 4.9; 7.2; 8.6 No. 92 Goat anti-mouse IgGCompound 1.35; 1.87; 2.69; 3.49; 4.20 No. 96

The fluorescence of the conjugates was measured using a JACSOfluorescence spectrophotometer and was then plotted against the DOL togive FIGS. 4-7.

Example 98 Preparation of a Phalloidin Dye-Conjugate

To aminophalloidin (1 mg) and compound Nos. 5, 7 and 23 (1.5equivalents) in DMF (200 μL) was added N,N-diisopropylethylamine (3equivalents) and the mixture was stirred at room temperature overnight.The solution was concentrated to dryness under vacuum and the residuewas purified by column chromatography by LH-20 column (1.5 mg). Theproduct is an effective stain for F-actin filaments in fixed-cellpreparations.

Example 99 Preparation and Use of a Fluorescent α-BungarotoxinDye-Conjugate

To a solution of α-bungarotoxin (1 mg) in 0.1 M sodium bicarbonate (25μL) was added compound No. 7 (1.5 equivalents) in one portion and themixture was stirred at room temperature for 2 hours. The product waspurified by G-25 size exclusion column and then by reverse-phase HPLC.Staining of acetylcholine receptors and detection of their resultingfluorescence, although detected at longer wavelength, was comparable tothat obtained with Texas Red α-bungarotoxin conjugate.

Example 100 Preparation of Aminodextran-Dye Conjugate

To a solution of 70000 MW aminodextran with an average of 13 aminogroups in 0.1M sodium bicarbonate (400 μL) is added compound No. 7 so asto give a dye/dextran of about 12. After 6 hours the conjugate ispurified on SEPHADEX G-50 with water as eluent. Typically 4-6 moles ofdye are conjugated to 70000 MW dextran.

Example 101 Preparation of Dye-Bacteria Conjugates

Heat killed Escherichia coli are suspended in pH 8-9 buffer (10 mg/mL)and then incubated with 0.5-1.0 mg/mL of an amine-reactive dye such ascompound No. 7. After 30-60 minutes the labeled bacteria are centrifugedand washed several times with buffer to remove any free dye. The labeledbacteria is analyzed by flow cytometry.

Example 102 Preparation of Nucleotide-Dye Conjugates

To a solution of 5-(3-aminoallyl)-2-deoxyuridine 5′-triphosphate (2 mg,Sigma Chemical) in H₂O (100 μL) is added compound No. 7 or compound No.23 in DMF and triethylamine (5 μL). The mixture is stirred at roomtemperature for 3 hours and then concentrated to dryness in vacuo. Theresidue is purified by preparative HPLC. The product fractions arelyophilized to give a dark blue nucleotide conjugate. Alternatively,fluorescent dye-conjugates of deoxyuridine 5′-triphosphate are preparedfrom 5-(3-amino-1-propynyl)-2′-deoxyuridine 5′-triphosphate or bytreating a thiolated nucleotide or a thiophosphate nucleotide with athiol reactive dye of the invention such as compound No. 85.

Additionally, 2′-(or 3′)-2-aminoethylaminocarbonyladenosine5′-triphosphate is reacted with slight excess of compound No. 7 andfollowing the precipitation with ethanol, the ribose-modified product ispurified by preparative HPLC. Additional nucleotides conjugates with thedyes of invention can readily prepared by someone skilled in the artfollowing the published procedures such as Nimmakayalu M. et al.,Biotechniques, 2000, 28, 518; Muhlegger K. et al., Biol. Chem. HoppeSeyler, 1990, 371, 953; Giaid A. et al. Histochemistry, 1989, 93, 191.

Example 103 Preparation of an Oligonucleotide Dye-Conjugate

To a 5′-amine-modified, 18-base M13 primer sequence (100 μg) in H₂O (4μL) is added a solution of compound No. 7 (500 μg) in 0.1M sodium boratepH=8.5 buffer (200 μL). The mixture is stirred at room temperatureovernight and 3 volumes of cold ethanol are added. The mixture is cooledto −20° C., centrifuged, the supernatant is decanted, the pellet isrinsed with ethanol and then dissolved in H₂O (100 μL). The labeledoligonucleotide is purified by preparative HPLC. The desired peak iscollected and evaporated to give the fluorescent oligonucleotide.

Example 104 Flow Cytometry Analysis of Cells Extracellularly Stainedwith Dye-Antibody Conjugates

One million Jurkat cells per sample were stained with 0.25 μg mouseanti-human CD3 (BD Biosciences) followed by 1 μg of goat anti-mouse IgGlabeled with compound No. 41 at the DOL shown in FIG. 8 (Example 96).Flow cytometry was performed on a Beckman Coulter FC-500 using CXPsoftware. Noise represents average fluorescence intensity from cellsstained with only goat anti-mouse-compound No. 41 conjugate asbackground control. Signal represents average fluorescence intensityfrom cells stained with CD3 and goat anti-mouse-compound No. 41.

Example 105 Flow Cytometry Analysis of Cells Intracellularly Stainedwith Dye-Antibody Conjugates

One million Jurkat cells were fixed, permeabilized, and incubated with0.25 μg mouse anti-human CD3 antibody (BD Biosciences). The CD3 antibodywas followed by incubation with 1 μg goat anti-mouse IgG conjugatedAlexaFluor647 (DOL 3.1) or compound No. 41 (DOL 3.9) (Example 96). About10,000 cells from each sample were analyzed on a BD FACS Calibur flowcytometer and fluorescence was detected in the FL4 channel.

Example 106 Labeling β-Galactosidase with a Thiol Reactive Dye

A solution of β-galactosidase, a protein rich in free thiol groups, isprepared in PBS buffer (1 mg in 200 μL) and then treated with a solutionof thiol reactive compound No. 85 (5 mg) in DMF (100 μL). Unreactive dyeis removed by centrifugation using Nanosep centrifugal device. Thedegree of substitution by the dye is estimated using the method cited inExample 96.

Example 107 Photostability Comparison Among Compound No. 96 Alexa Fluor488 and Fluorescein

Actin filaments were stained with phalloidin labeled with compound No.96, Alexa Fluor 488 or fluorescein (the phalloidin conjugates wereprepared from aminophalloidin and the succinimidyl ester form of thedyes using the procedure described in Example 98). After washing, eachstained sample was continuously illuminated and the fluorescenceintensity was monitored by taking measurement every 5 seconds. Therelative fluorescence vs. time for each sample was plotted (FIG. 11).For a long time, fluorescein had been the dye of choice for greenfluorescence color because the dye's absorption peak well matches withthe 488 nm argon laser line. However, fluorescein undergoesphotobleaching very quickly, limiting its use for microscopy studies.Alexa Fluor 488 from Molecular Probes, Inc. was developed as a superioralternative because of its exceptional photostability. FIG. 11demonstrates that compound No. 96 of the invention is more photostablethan Alexa Fluor 488.

Example 108 Measuring the Serum Half-Life of a Labeled Biomolecule ofthe Invention

The in vivo serum half-life of a labeled biomolecule of the inventionmay be measured, for example, after injection of a labeled biomoleculeinto catheterized rats, for example as described by [Pepinsky, R. B., etal. (2001) J Pharmacol Exp Ther, 297: 1059-66]. The plasma concentrationof the biomolecule is then measured in extracted blood samples. Suchsamples may be withdrawn at various time points depending on the timecourse studied. The concentration of the labeled biomolecule may bemeasured using a variety of methods, including fluorescence measurementsand/or biochemical techniques such as ELISA or Western Blots. Thestabilizing effect of a fluorescent group of the invention may bemeasured by comparing the stability of the labeled biomolecule of theinvention relative to a corresponding biomolecule lacking said dye.

Example 109 Measurement and Comparison of Aggregation Behavior of anAntibody Labeled with a Dye of the Invention and the Same AntibodyLabeled with Other Commercially Available Dyes

A set of goat anti-mouse IgG conjugates were prepared by labeling aportion of goat anti-mouse IgG with one of three near IR dyes.Additionally for each dye, separate portions were labeled at one ofseveral different degree of labeling (DOL), to observe the absorptionspectra for aggregation behavior as the concentration of dye moleculeson the antibody is increased with increased degree of labeling. The dyesutilized were Cy7® dye, Alexa Fluor 750® (AF750®) dye, and a dye of theinvention, Dye No. 29 (Table 3). FIG. 12A shows the absorption spectraof the conjugate formed from labeling goat anti-mouse IgG with Cy7® dyeat 4 different DOL (1.2 to 3.4 dye molecules per antibody). FIG. 12Bshows the absorption spectra of the conjugate formed from labeling goatanti-mouse IgG with Alexa Fluor 750® (AF750®) dye at four different DOL(2.2 to 5.9 dye molecules per antibody). FIG. 12C shows the absorptionspectra of the conjugate formed from labeling goat anti-mouse IgG withDye No. 29 (Table 3), at six different DOL (1.1 to 7.4 dye molecules perantibody). All spectra were taken at room temperature in PBS 7.4 buffer.The spectra of both Cy7® dye- and AF750® dye-labeled conjugates (A andB) display a double peak characteristic of dye aggregation while thespectra of Compound No. 29-labeled conjugates (C) show substantially asingle peak, indicating a substantial lack of dye aggregation.

Example 110 Measurement and Comparison of Fluorescent Signal of anAntibody Labeled with a Dye of the Invention and the Same AntibodyLabeled with Other Commercially Available Dyes which Lack a WaterSoluble Polymer Group

A set of goat anti-mouse IgG conjugates were prepared by labeling aportion of goat anti-mouse IgG with one of three near IR dyes.Additionally for each dye, separate portions were labeled at one ofseveral different degree of labeling (DOL), to observe the fluorescentoutput as the concentration of dye molecules on the antibody isincreased with increased degree of labeling. The dyes utilized were: adye of the invention, Dye No. 29 (Table 3) Cy7® dye, and Alexa Fluor750® (AF750®) dye. The Cy7® dye and Alexa Fluor 750® (AF750®) dye do nothave a water soluble polymer group. FIG. 13 shows total fluorescence vs.degree of labeling (DOL) for Dye 29, Alexa Fluor 750® (AF750®) dye andCy7® dye, at identical protein concentrations in pH 7.4 PBS buffer, whenexcited at 735 nm. The data shows that, compared to AF750® dye and Cy7®dye, the fluorescent group of Dye 29 has higher fluorescence quantumyield over a wide degree of labeling and has less fluorescence quenchingwhen the antibody is at higher degrees of labeling.

Example 111 Measurement and Comparison of the Fluorescent Signal Arisingfrom Intracellular Staining of Jurkat Cells with Goat Anti-Mouse IgGLabeled with Three Near-IR Dyes

A set of goat anti-mouse IgG labeled conjugates were prepared fromlabeling a portion of goat anti-mouse IgG with one of Dye No. 29 (Table3), Alexa Fluor 750® dye or Cy7® dye. Jurkat cells were first labeledwith mouse anti-human CD3 antibody and then stained with one of thethree labeled secondary antibodies, which all have a similar degree oflabeling. To measure the background fluorescence from each labeledsecondary antibody, the cells were also stained directly with each ofthe fluorescent secondary antibody without the primary antibody (darkcolumns). FIG. 14 shows the relative fluorescence levels of the Jurkatcells stained with goat anti-mouse IgG labeled with Dye No. 29 (Table3), Alexa Fluor 750® dye and Cy7® dye, respectively, as measured by flowcytometry. The results demonstrate that cells stained with antibodyconjugate of this invention are significantly brighter and haveexcellent signal-to-noise ratio.

Example 112 Determination and Comparison of Photostability for ThreeNear IR Dyes

FIG. 15 compares the photostability of three near-IR dyes: Compound No.29 of Table 3, Alexa Fluor 750® (AF750®) dye and Cy7® dye. Solutions ofthe three dyes at 5 μM dye concentration were exposed to sun light for ½hour. Absorption spectra of the solutions were recorded before and afterthe photolysis. The results show the near-IR dye of the invention issignificantly more stable than both AF750® dye and Cy7® dye.

Example 113 Measurement and Comparison of the Fluorescent Signal Arisingfrom Intracellular Staining of Jurkat Cells with Goat Anti-Mouse IgGLabeled with APC-Alexa Fluor 750® Tandem Dye or Dye 29 (Table 3)

FIGS. 16A and B show the relative fluorescence levels of Jurkat cellsstained intracellularly with an antibody labeled with APC-Alexa Fluor750® tandem dye or Dye 29 (Table 3). FIG. 16A is a graphicalrepresentation showing the relative fluorescence levels of Jurkat cellsstained with indicated amount of either goat anti-mouse IgG labeled withcompound No. 29 (DOL=3.5) or a commercially available goat anti-mouseIgG labeled with an APC-AF750® tandem dye (Invitrogen), as measured byflow cytometry. The cells were first labeled with mouse anti-human CD3antibody and then stained with one of the two labeled secondaryantibodies. To measure the background fluorescence from each labeledsecondary antibody, the cells were also stained directly with thefluorescent secondary antibody without the primary antibody (darkenedcolumns). FIG. 16B shows the signal-to-noise ratios of the stainingsfrom FIG. 16A. Flow cytometry experiments were performed on a BD LSR IIequipped with a 633 nm laser and 780/60 nm PMT detector. The resultsdemonstrate that Dye No. 29 gives excellent fluorescent signal with verylittle background while the APC-AF750® tandem dye showed significantnonspecific staining. Dye No. 29 has very little absorption at 633 nmwhereas the tandem dye has nearly maximal absorption at the laserwavelength due to the donor dye APC. Because tandem dyes such asAPC-AF750® dyes are much more difficult and thus expensive tomanufacture than a simple dye such as Dye No. 29, near-IR dyes of theinvention are particularly advantageous for flow cytometry analysisusing a 633 nm or longer wavelength excitation source.

Example 114 Determination of Total Fluorescence as a Function of Degreeof Labeling for Goat Anti-Mouse IgG Conjugates of Three Near-IR Dyeswith Similar Wavelengths

FIG. 17 shows the total fluorescence vs. degree of labeling (DOL) forgoat anti-mouse IgG conjugates of a dye of the invention, Dye No. 31(Table 3), Dylight™ 800 Dyefrom Thermo Fisher, and IRDye 800® CW dyefrom Li-Cor Biosciences, all of which have similar wavelengths. The datashow that Dye No. 31 is significantly brighter than the other two dyesover a wide range of DOL.

Example 115 Determination of Fluorescent Signal as a Function of Degreeof Labeling and Comparison of Fluorescent Quenching and Signal-to-Noisefor Goat Anti-Mouse IgG Conjugates of Three Near IR Dyes

FIG. 18 shows the relative fluorescence levels of Jurkat cells stainedwith goat anti-mouse IgG antibodies labeled with Dye No. 31 (Table 3),Dylight™ 800 dye from Thermo Fisher and IRDye 800® CW dye from Li-CorBiosciences, respectively, as measured by flow cytometry. To assess thefluorescence quenching of the three near-IR dyes, the antibody waslabeled with each dye at several different degrees of labeling (DOL) asindicated. The cells were first labeled with mouse anti-human CD3antibody and then stained with one of the labeled secondary antibodies.To measure the background fluorescence from each labeled secondaryantibody, the cells were also stained directly with each of thefluorescent secondary antibodies without the primary antibody (isotype,dark columns). The results show that Dye No. 31 is significantlybrighter than both Dylight™ 800 dye and IRDye 800® CW dye over a widerange of DOL. Also importantly, Dye No. 31 produced much bettersignal-to-noise ratio than the other two dyes.

Example 116 Measurement and Comparison of Fluorescence Quantum Yield forFour Spectrally Similar Near IR Dyes

FIG. 19 is a plot of total fluorescence vs. degree of labeling (DOL) forgoat anti-mouse IgG conjugates of a near-IR dye of the invention, DyeNo. 32 (Table 3), and three spectrally similar near-IR fluorescentgroups, Cy5.5® dye from GE Healthcare, Alexa Fluor 680® (AF680) dye fromInvitrogen and Dylight™ 680 from Thermo Fisher, respectively.Fluorescence measurements were made in pH 7.4 PBS buffer using 660 nmexcitation. The data shows that, compared to Cy5.5®, Alexa Fluor 680®and Dylight™ 680, the fluorescent group of the invention has higherfluorescence quantum yield over a wide degree of labeling.

Example 117 Measurement and Comparison of Relative Fluorescent Signalfrom Jurkat Cells Intracellularly Stained by Goat Anti-Mouse IgGAntibodies Labeled Individually with Four Different Near IR Dyes

FIGS. 20A and B show data related to the relative fluorescence level ofJurkat cells stained with goat anti-mouse IgG antibodies labeledindividually with four different near IR dyes. FIG. 20A shows therelative fluorescence levels of Jurkat cells stained with goatanti-mouse IgG antibodies labeled with Dye No. 32 (Table 3), Cy5.5® dyefrom GE Healthcare, Dylight™ 680 dye from Thermo Fisher or Alexa Fluor680® (AF680®) dye from Invitrogen, as measured by flow cytometry. Toassess the fluorescence quenching of the three near-IR dyes, eachportion of goat anti-mouse IgG antibody was labeled with one dye at oneof two different degree of labeling (DOL) as indicated to form a set ofeight antibodies. The cells were first labeled with mouse anti-human CD3antibody and then stained with one of the labeled secondary antibodies.To measure the background fluorescence from each labeled secondaryantibody, the cells were also stained directly with each of thefluorescent secondary antibodies without the primary antibody (isotype,dark columns). FIB. 20B is a plot of signal-to-noise ratio (S/N) for thestaining results in FIG. 20A. The data show that conjugates labeled withDye No. 32 are much brighter and more specific in staining thanconjugates prepared from the other three commercial near-IR dyes.

Example 118 Preparation of Dye 29

Dye 29 was prepared in 4 steps from common intermediates used inprevious examples herein. Compound No. 19 (84 mg), compound No. 59, andsodium acetate (4 equivalents) were combined in a mixture of acetic acid(2 mL) and acetic anhydride (1 mL). The resulting mixture is heated at120° C. for 20 min to form the intermediate compound No. 29a.

The solvent was distilled off under vacuum. The product was purified bySephadex LH-20 using water as the eluent. Compound No. 29a (30 mg) wasconverted to the peggylated intermediate compound No. 29b by followingthe procedure to make compound No. 21 (Example 21). The methyl esterintermediate compound No. 21b (18 mg) was hydrolyzed to the free acidcompound No. 29c by following the procedure to make compound No. 22(Example 22). Compound No. 29c (5 mg) was activated to the final productDye 29 using TSTU and triethylamine as described for compound No. 23 inExample 23.

Example 118 Preparation of Dye 31

Dye 31 was prepared in 4 steps from common intermediates used inprevious examples herein. Compound No. 19 (12 g) was first treated withglutoconaldehyde dianil hydrochloride (18 g) and Et₃N (0.5 mL) in AcOH(40 mL) at 120° C. for 3 hours. After cooling down to room temperature,the mixture was concentrated to dryness under vacuum and the residue waspurified by column chromatography on silica gel to give compound No. 31aas a red brown gummy solid (3 g).

The above intermediate (1 g) was coupled toN-(5-carboxypentyl)-1,3,3-trimethylbenzindolenium-6,8-dilsulfonate (1equivalent) (Bioconjugate Chem. 7, 356 (1996)) to give compound No. 31b(80 mg) using the condition to make compound No. 29a in Example 109.

Compound No. 31b (25 mg) was converted to compound No. 31c (60 mg),followed by hydrolysis to the free acid compound No. 31d using theprocedures described in Example 109. Finally, the free acid dye compoundNo. 31d (10 mg) was activated to Dye 31 using TSTU and triethylamine(Example 109).

Example 120 Preparation of Dye 32

Dye 32 was prepared using sulfonated benzindolium intermediates andprocedures similar to those used for preparing compound No. 23 (Example23). Briefly,N-(5-carboxypentyl)-1,3,3-trimethylbenzindolenium-6,8-dilsulfonate(Example 110) (5 g) was reacted with malonaldehyde dianil hydrochloride(5 g), Et₃N (0.5 mL) in AcOH (50 mL) was heated at 120° C. for 3 hours.After cooling down to room temperature, the mixture was concentrated todryness under vacuum and the residue was purified by columnchromatography on silica gel to compound No. 32a as an orange red gummysolid (2.5 g).

Compound No. 62 (Example 62) (5 g) was thoroughly mixed with6-bromohexanoic acid (1.5 equivalent) and then heated at 95° C. for 24 hto form the benzindolium intermediate compound No. 32b:

Compound No. 32a and compound No. 32b were coupled to form the cyaninemethyl ester intermediate compound No. 32c, which is subsequentlypeggylated to form compound No. 32d, followed by hydrolysis to form thefree acid compound No. 32e and final conversion to the succinimidylester Dye 32.

Procedures for the steps leading from compound No. 32c to compound No.32e are analogous to those used for preparing compound No. 23 (Example23).

What is claimed is:
 1. A compound of any one of the formulae:


2. A kit comprising: i) the compound of claim 1; ii) a buffer; iii)materials or devices for purifying conjugation products; and iv)instructions instructing the use of the compound.
 3. A biomoleculehaving an amine, a thiol, a hydroxyl, or an aldehyde functional groupcomprising a label having a structure of a Formula of claim 1, whereinthe at least one reactive moiety of the Formula has undergone a reactionwhich attaches the label to the biomolecule.
 4. The biomoleculecomprising a label of claim 3 wherein the biomolecule comprises apolynucleotide.
 5. The biomolecule comprising a label of claim 3 whereinthe biomolecule comprises a polypeptide.
 6. The biomolecule of claim 5,wherein the polypeptide further comprises an antigen binding site. 7.The biomolecule of claim 5, wherein the polypeptide is a wholeimmunoglobulin.
 8. The biomolecule of claim 5, wherein the polypeptideis a Fab fragment.
 9. An immunoglobulin having an amine, a thiol, ahydroxyl, or an aldehyde functional group labeled with a compound ofclaim
 1. 10. The immunoglobulin of claim 9, wherein the immunoglobulinretains binding specificity to a target upon conjugation to thefluorescent compound.
 11. The immunoglobulin of claim 10, wherein theimmunoglobin is an antibody that binds specifically to an antigen on acancer cell.
 12. The immunoglobin of claim 11 wherein the antibody bindsto erb2.
 13. An immunoglobin having an amine, a thiol, a hydroxyl, or analdehyde functional group comprising a label having a structure of aFormula of claim 1 wherein the at least one reactive moiety of theFormula has undergone a reaction which attaches the label to theimmunoglobin, wherein the immunoglobin is an antibody that bindsspecifically to an antigen on a cancer cell.
 14. The immunoglobin ofclaim 13 wherein the antibody binds to erb2.
 15. A polypeptide having anamine, a thiol, a hydroxyl, or an aldehyde functional group labeled witha fluorescent compound, the polypeptide exhibiting a serum half-life noshorter than that of a corresponding polypeptide that lacks thefluorescent compound, wherein the fluorescent compound is a compound ofclaim
 1. 16. A method of preparing a labeled biomolecule comprisingreacting a compound of claim 1 and a substrate biomolecule underconditions sufficient to effect crosslinking between the compound andthe substrate biomolecule.
 17. The method of claim 16, wherein thesubstrate biomolecule is a polypeptide, a polynucleotide, acarbohydrate, a lipid or a combination thereof.
 18. The method of claim17, wherein the substrate biomolecule is a polynucleotide.
 19. A methodof labeling a polypeptide comprising: forming a complex that comprisesthe polypeptide and a binding agent, wherein the binding agent comprisesa fluorescent label having a structure of formula of claim 1 wherein theat least one reactive moiety of the Formula has undergone a reactionwhich attaches the label to the binding agent.
 20. The method of claim19 wherein the binding agent is an antibody.
 21. The method of claim 20wherein the complex comprises (a) a primary antibody that binds to thepolypeptide, and (b) the binding agent which functions as a secondaryantibody exhibiting binding capability to the primary antibody.
 22. Themethod of claim 21, wherein the labeling occurs on a solid substrate.23. The method of claim 21 that labels a polypeptide intracellularly.24. The method of claim 21, wherein the complex yields a signal to noiseratio greater than about 100, wherein the signal to noise ratio iscalculated by the formula:(fluorescent signal from a complex comprising the polypeptide bound by aprimary antibody which in turn is bound to the bindingagent)/(fluorescent signal from a mixture of the polypeptide, an isotypecontrol primary antibody and the binding agent).
 25. The method of claim21, wherein the complex yields a signal to noise ratio greater thanabout 250, wherein the signal to noise ratio is calculated by theformula:(fluorescent signal from a complex comprising the polypeptide bound by aprimary antibody which in turn is bound to the bindingagent)/(fluorescent signal from a mixture of the polypeptide, an isotypecontrol primary antibody and the binding agent).
 26. The method of claim21, wherein the complex yields a signal to noise ratio greater thanabout 270, wherein the signal to noise ratio is calculated by theformula:(fluorescent signal from a complex comprising the polypeptide bound by aprimary antibody which in turn is bound to the bindingagent)/(fluorescent signal from a mixture of the polypeptide, an isotypecontrol primary antibody and the binding agent).
 27. A method forlabeling a cell within a population of cells whereby the cell isdifferentially labeled relative to neighboring cells within thepopulation, the method comprising contacting the cell with a biomoleculeof claim 3, wherein the biomolecule comprises a targeting moiety thatbinds to a binding partner that is indicative of the cell, and therebydifferentially labeling the cell relative to neighboring cells withinthe population.
 28. The method of claim 27, further comprising the stepof imaging the cell, the imaging step comprising: i) directing excitingwavelength to the cell; and ii) detecting emitted fluorescence from thecell.
 29. The method of claim 27, wherein the labeling takes place invitro.
 30. The method of claim 27, wherein the labeling takes place invivo.